Small molecule inhibitors of the nuclear translocation of androgen receptor for the treatment of castration-resistant prostate cancer

ABSTRACT

wherein R20 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy, aryloxy, a thio-containing group, or a seleno-containing group; Z is alkanediyl, substituted alkanediyl, cycloalkanediyl, or substituted cycloalkanediyl; Y is S, O, S(═O), —S(═O)(═O)—, or NR10, wherein R10 is H or alkyl; R21 is alkanediyl, substituted alkanediyl, cycloalkanediyl, substituted cycloalkanediyl alkadienyl, substituted alkadienyl, alkatrienyl, substituted alkatrienyl; X is —C(═O)—, —S(═O)(═O)—, or —N(H)C(═O)—; R22 includes at least one divalent amino radical; R23 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy, aryloxy, a thio-containing group, or a seleno-containing group; a, b, c, and d independently are 0 or 1.

This application is a divisional of U.S. application Ser. No.15/080,237, filed Mar. 24, 2016, which is a continuation-in-part of U.S.application Ser. No. 15/023,349, filed Mar. 18, 2016, which is the U.S.National Stage of International Application No. PCT/US2014/056369, filedSep. 18, 2014, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.61/880,747, filed Sep. 20, 2013, each of which is incorporated in itsentirety herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant #GM067082awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Castration-resistant prostate cancer (CRPC) is currently incurable andmakes prostate cancer the second most common cause of cancer death amongmen in the United States. The androgen receptor (AR) is activated viamultiple mechanisms including AR overexpression, mutation,hypersensitization, and/or intratumoral androgen synthesis in patientsrelapsed after androgen deprivation therapy (ADT). The steroidalhormones testosterone and dihydrotestosterone are the major endogenousandrogens that cause nuclear translocation and subsequent activation ofandrogen receptor (AR). Overexpression and knockdown studies havedemonstrated that AR is a key molecular determinant and an excellenttherapeutic target for CRPC. Clinical use of abiraterone, a potentinhibitor of testosterone synthesis, and MDV3100 (enzalutamide) andbicalutamide, AR antagonists, indicates that AR remains a viable targetin a significant number of CRPC patients.

Androgen receptor (AR), a member of the steroid receptor superfamily, isa ligand-dependent transcription factor that controls the expression ofandrogen-responsive genes. Intracellular trafficking is an importantmechanism in the regulation of many transcription factors, including AR.In order to access its target genes, a transcription factor requireslocalization to the nucleus. Retention of a transcription factor in thecytoplasm prevents its activity. Thus, a key regulatory step in theaction of AR is its nuclear translocation. In androgen-sensitive cells,AR is localized to the cytoplasm in the absence of ligand. Upon additionof androgens, AR translocates to the nucleus and transactivates targetgenes. However, in CRPC cells, AR remains in the nucleus even in theabsence of androgen and transactivates androgen-responsive genes,leading to uncontrolled growth of prostate tumors. Therefore, novelapproaches that can block the nuclear localization of AR, degradenuclear AR, and/or suppress nuclear AR activity may provide an effectivetherapy against CRPC.

SUMMARY

Disclosed herein is a compound, or a pharmaceutically acceptable salt orester thereof, having a formula I of:

R²⁰—(Z)_(b)—(Y)_(c)—(R²¹)_(a)—(X)_(d)—R²²—R²³

wherein R²⁰ is an aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy,aryloxy, amino, a thio-containing group, or a seleno-containing group; Zis alkanediyl, substituted alkanediyl, cycloalkanediyl, or substitutedcycloalkanediyl; Y is S, O, S(═O), —S(═O)(═O)—, or NR¹⁰, wherein R¹⁰ isH or alkyl; R²¹ is alkanediyl, substituted alkanediyl, cycloalkanediyl,substituted cycloalkanediyl alkadienyl, substituted alkadienyl,alkatrienyl, substituted alkatrienyl; X is —C(═O)—, —S(═O)(═O)—, or—N(H)C(═O)—; R²² is a moiety that includes at least one divalent aminoradical; R²³ is an aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy,aryloxy, amino, a thio-containing group, a seleno-containing group; a is0 or 1; b is 0 or 1; c is 0 or 1; and d is 0 or 1. In some embodiments,if X is —C(═O)— then Y is not S. In certain embodiments, R²¹ iscycloalkanediyl. When R²¹ is cycloalkanediyl, R²⁰ may be a phenyloptionally substituted with at least one halogen and/or R²³ may be aphenyl substituted with at least one halogen and/or at least one alkyl.

Also disclosed herein is a method for treating prostate cancer in asubject, comprising administering a therapeutically effective amount ofan agent to the subject, wherein the agent is a compound, or apharmaceutically acceptable salt or ester thereof, of formula I orformula II.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D are a table showing compound structures.

FIGS. 2A through 2E show assay results for several of the compounds.C4-2 cells were transfected with PSA6.1-Luc, GFP-AR, and pRL-CMV andthen treated with indicated doses for 24 hours. For luciferase assays,cells were lysed with passive lysis buffer (Promega) and both Fireflyand Renilla luciferase activities were read using a Dual-LuciferaseReporter Assay kit (Promega) on a LmaxII384 luminometer (MolecularDevices). Firefly luciferase values were normalized to Renilla(pRL-CMV). Plotted values represent averaged normalized Fireflyluciferase activities, each performed in triplicate, relative to DMSOcontrol. This assay is described in more detail in PCT PatentApplication Publication WO 2013055793, which is incorporated herein byreference.

FIG. 3 is a reaction scheme showing the synthesis of2-((isoxazol-4-ylmethyl)thio)-1-(4-phenylpiperazin-1-yl)ethanone 1.

FIG. 4 is a chemical structure of2-((isoxazol-4-ylmethyl)thio)-1-(4-phenylpiperazin-1-yl)ethanone showingzones of modification.

FIG. 5 is a reaction scheme showing synthesis of certain embodiments ofthe disclosed compounds. Reagents and conditions: (a) T3P(propylphosphonic anhydride), Et₃N (triethylamine), CH₂Cl₂, rt (roomtemperature), overnight, 52-98%; (b) LiAlH₄, dry THF (tetrahydrofuran),0° C., 1 h, 42%; (c) NaIO₄, MeOH (methanol), H₂O, rt, 15 h, 68%; (d)m-CPBA (meta-chloroperoxybenzoic acid), CH₂Cl₂, rt, 15 h, 44%.

FIG. 6 is a reaction scheme showing synthesis of certain embodiments ofthe disclosed compounds. Reagents and conditions: (a) T3P, Et₃N, CH₂Cl₂,rt, overnight, 62-96%; (b) Lindlar's catalyst, quinoline, H₂, EtOAc(ethyl acetate), quant.; (c) CrCl₂, CH₂ICl, THF, reflux, overnight, 57%.

FIG. 7 is a reaction scheme showing synthesis of certain embodiments ofthe disclosed compounds. Reagents and conditions: (a) 2-chloroacetylchloride, Et₃N, CH₂Cl₂, rt, overnight, 99%; (b) chloromethanesulfonylchloride, Et₃N, CH₂Cl₂, rt, overnight, 85%; (c) NaH, THF, rt, 1-2 d,4-99%; (d) DPPA (diphenyl phosphoryl azide), Et₃N, toluene, reflux,overnight, 17-65%.

FIG. 8 is a reaction scheme showing synthesis of certain embodiments ofthe disclosed compounds. Reagents and conditions: (a) Boo:), DMAP,CH₂Cl₂, rt, overnight, 78%; (b) NaHMDS (sodiumbis(trimethylsilyl)amide), PhNTf₂(N-phenyl-bis(tifluoromethanesulfonamide), THF, −78° C. to rt, 4 h, 78%;(c) Pd(PPh₃)₄, LiCl, Na₂CO₃, (2-Me)PhB(OH)₂, DME (dimethoxyethane), H₂O,60° C., 3 h, 78%; (d) H₂, Pd/C, EtOH (ethanol), rt, 14 h, 90%; (e) TFA(trifluoroacetic acid), CH₂Cl₂, rt, 16 h, quant.; (f) 2-chloroacetylchloride, Et₃N, THF, rt, 22 h, 79%; (g) 25, NaH, THF, rt, 1 d, 30%.

FIG. 9A is a graph showing the effect of compound #583 at indicatedconcentrations on PSA-driven luciferase activity in C4-2 cells.

FIG. 9B shows the effect of compound #583 at indicated concentrations onC4-2 cell proliferation in BrdU assay.

FIG. 9C shows the effect of analog #583 at indicated concentrations onPC3 cell proliferation in BrdU assay.

FIG. 10 is a graph showing the effect of compound #571 at indicatedconcentrations on PSA-driven luciferase activity in C4-2 cells.

FIG. 11 is a graph showing the effect of compound JJ-450 at indicatedconcentrations on 22Rv 1 xenograft tumor volume. JJ-450 was injectedi.p. daily.

FIG. 12 is a graph showing the effect of compound JJ-450 at indicatedconcentrations and administration route on LNCaP xenograft tumor volume.JJ-450 was administered 6 times, from Monday to Saturday, every week

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

“Administration of” and “administering a” compound should be understoodto mean providing a compound, a prodrug of a compound, or apharmaceutical composition as described herein. The compound orcomposition can be administered by another person to the subject (e.g.,intravenously) or it can be self-administered by the subject (e.g.,tablets).

“Alkanediyl” or “cycloalkanediyl” refers to a divalent radical of thegeneral formula —C_(n)H_(2n)— derived from aliphatic or cycloaliphatichydrocarbons.

The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl,halogenated alkyl and cycloalkyl groups as described above. A “loweraliphatic” group is a branched or unbranched aliphatic group having from1 to 10 carbon atoms.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A“lower alkyl” group is a saturated branched or unbranched hydrocarbonhaving from 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4carbon atoms. Alkyl groups may be “substituted alkyls” wherein one ormore hydrogen atoms are substituted with a substituent such as halogen,cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. Forexample, a lower alkyl or (C₁-C₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can be cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;(C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

The term “alkylaryl” refers to a group in which an alkyl group issubstituted for a hydrogen atom of an aryl group. An example is —Ar—R,wherein Ar is an arylene group and R is an alkyl group.

The term “alkoxy” refers to a straight, branched or cyclic hydrocarbonconfiguration and combinations thereof, including from 1 to 20 carbonatoms, preferably from 1 to 8 carbon atoms (referred to as a “loweralkoxy”), more preferably from 1 to 4 carbon atoms, that include anoxygen atom at the point of attachment. An example of an “alkoxy group”is represented by the formula —OR, where R can be an alkyl group,optionally substituted with an alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, alkoxy or heterocycloalkyl group.Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy,n-butoxy, i-butoxy, sec-butoxy, tert-butoxy cyclopropoxy, cyclohexyloxy,and the like.

“Alkoxycarbonyl” refers to an alkoxy substituted carbonyl radical,—C(O)OR, wherein R represents an optionally substituted alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety.

“Alkynyl” refers to a cyclic, branched or straight chain groupcontaining only carbon and hydrogen, and unless otherwise mentionedtypically contains one to twelve carbon atoms, and contains one or moretriple bonds. Alkynyl groups may be unsubstituted or substituted. “Loweralkynyl” groups are those that contain one to six carbon atoms.

The term “amide” or “amido” is represented by the formula —C(O)NRR′,where R and R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl,aryl, arylalkyl, cycloalkyl, halogenated alkyl, or heterocycloalkylgroup described above. A suitable amido group is acetamido.

The term “amine” or “amino” refers to a group of the formula —NRR′,where R and R′ can be, independently, hydrogen or an alkyl, alkenyl,alkynyl, aryl, arylalkyl, carbonyl (e.g, —C(O)R″, where R″ can behydrogen, an alkyl, alkenyl, alkynyl, aryl, or an arylalkyl),cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example,an “alkylamino” or “alkylated amino” refers to —NRR′, wherein at leastone of R or R′ is an alkyl.

“Aminocarbonyl” alone or in combination, means an amino substitutedcarbonyl (carbamoyl) radical, wherein the amino radical may optionallybe mono- or di-substituted, such as with alkyl, aryl, arylalkyl,cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyland the like. An aminocarbonyl group may be —C(O)—N(R) (wherein R is asubstituted group or H). An “aminocarbonyl” is inclusive of an amidogroup. A suitable aminocarbonyl group is acetamido.

An “analog” is a molecule that differs in chemical structure from aparent compound, for example a homolog (differing by an increment in thechemical structure or mass, such as a difference in the length of analkyl chain or the inclusion of one of more isotopes), a molecularfragment, a structure that differs by one or more functional groups, ora change in ionization. An analog is not necessarily synthesized fromthe parent compound. Structural analogs are often found usingquantitative structure activity relationships (QSAR), with techniquessuch as those disclosed in Remington (The Science and Practice ofPharmacology, 19th Edition (1995), chapter 28). A derivative is amolecule derived from the base structure.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats. The term subject appliesregardless of the stage in the organism's life-cycle. Thus, the termsubject applies to an organism in utero or in ovo, depending on theorganism (that is, whether the organism is a mammal or a bird, such as adomesticated or wild fowl).

The term “aryl” refers to any carbon-based aromatic group including, butnot limited to, phenyl, naphthyl, etc. The term “aryl” also includes“heteroaryl group,” which is defined as an aromatic group that has atleast one heteroatom incorporated within the ring of the aromatic group.Examples of heteroatoms include, but are not limited to, nitrogen,oxygen, sulfur, and phosphorous. The aryl group can be substituted withone or more groups including, but not limited to, alkyl, alkynyl,alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy,carboxylic acid, or alkoxy, or the aryl group can be unsubstituted.

The term “arylalkyl” refers to an alkyl group where at least onehydrogen atom is substituted by an aryl group. An example of anarylalkyl group is a benzyl group.

“Carbonyl” refers to a group of the formula —C(O)—. Carbonyl-containinggroups include any substituent containing a carbon-oxygen double bond(C═O), including acyl groups, amides, carboxy groups, esters, ureas,carbamates, carbonates and ketones and aldehydes, such as substituentsbased on —COR or —RCHO where R is an aliphatic, heteroaliphatic, alkyl,heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine

“Carboxyl” refers to a —COO group. Substituted carboxyl refers to —COORwhere R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or acarboxylic acid or ester.

The term “cycloalkyl” refers to a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. The term “heterocycloalkyl group” is acycloalkyl group as defined above where at least one of the carbon atomsof the ring is substituted with a heteroatom such as, but not limitedto, nitrogen, oxygen, sulfur, or phosphorous.

The term “co-administration” or “co-administering” refers toadministration of a first agent with a second agent within the samegeneral time period, and does not require administration at the sameexact moment in time (although co-administration is inclusive ofadministering at the same exact moment in time). Thus, co-administrationmay be on the same day or on different days, or in the same week or indifferent weeks. The first agent and the second agent may be included inthe same composition or they may each individually be included inseparate compositions. In certain embodiments, the two agents may beadministered during a time frame wherein their respective periods ofbiological activity overlap. Thus, the term includes sequential as wellas coextensive administration of two or more agents.

“Derivative” refers to a compound or portion of a compound that isderived from or is theoretically derivable from a parent compound.

The terms “halogenated alkyl” or “haloalkyl group” refer to an alkylgroup as defined above with one or more hydrogen atoms present on thesegroups substituted with a halogen (F, Cl, Br, I).

The term “hydroxyl” is represented by the formula —OH.

The term “hydroxyalkyl” refers to an alkyl group that has at least onehydrogen atom substituted with a hydroxyl group. The term “alkoxyalkylgroup” is defined as an alkyl group that has at least one hydrogen atomsubstituted with an alkoxy group described above.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

“N-heterocyclic” refers to mono or bicyclic rings or ring systems thatinclude at least one nitrogen heteroatom. The rings or ring systemsgenerally include 1 to 9 carbon atoms in addition to the heteroatom(s)and may be saturated, unsaturated or aromatic (includingpseudoaromatic). The term “pseudoaromatic” refers to a ring system whichis not strictly aromatic, but which is stabilized by means ofdelocalization of electrons and behaves in a similar manner to aromaticrings. Aromatic includes pseudoaromatic ring systems, such as pyrrolylrings.

Examples of 5-membered monocyclic N-heterocycles include pyrrolyl,H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including1,2,3 and 1,2,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl,isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls),tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), anddithiazolyl. Examples of 6-membered monocyclic N-heterocycles includepyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, and triazinyl. The heterocycles may beoptionally substituted with a broad range of substituents, andpreferably with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl,halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono ordi(C₁₋₆alkyl)amino The N-heterocyclic group may be fused to acarbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl,and anthracenyl.

Examples of 8, 9 and 10-membered bicyclic heterocycles include 1Hthieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl,benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl,indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl, and the like.These heterocycles may be optionally substituted, for example with C₁₋₆alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, hydroxy, mercapto,trifluoromethyl, amino, cyano or mono or di(C₁₋₆alkyl)amino Unlessotherwise defined optionally substituted N-heterocyclics includespyridinium salts and the N-oxide form of suitable ring nitrogens.

Examples of N-heterocycles also include bridged groups such as, forexample, azabicyclo (for example, azabicyclooctane).

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), arylalkyl (for example benzyl), aryloxyalkyl (forexample, phenoxymethyl), aryl (for example, phenyl, optionallysubstituted by, for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4alkoxy) or amino); sulphonate esters, such as alkyl- orarylalkylsulphonyl (for example, methanesulphonyl); or amino acid esters(for example, L-valyl or L-isoleucyl). A “pharmaceutically acceptableester” also includes inorganic esters such as mono-, di-, ortri-phosphate esters. In such esters, unless otherwise specified, anyalkyl moiety present advantageously contains from 1 to 18 carbon atoms,particularly from 1 to 6 carbon atoms, more particularly from 1 to 4carbon atoms. Any cycloalkyl moiety present in such estersadvantageously contains from 3 to 6 carbon atoms. Any aryl moietypresent in such esters advantageously comprises a phenyl group,optionally substituted as shown in the definition of carbocycylyl above.Pharmaceutically acceptable esters thus include C₁-C₂₂ fatty acidesters, such as acetyl, t-butyl or long chain straight or branchedunsaturated or omega-6 monounsaturated fatty acids such as palmoyl,stearoyl and the like. Alternative aryl or heteroaryl esters includebenzoyl, pyridylmethyloyl and the like any of which may be substituted,as defined in carbocyclyl above. Additional pharmaceutically acceptableesters include aliphatic L-amino acid esters such as leucyl, isoleucyland especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

It will be appreciated that the compounds described herein may havemetal binding, chelating, complex forming properties and therefore mayexist as metal complexes or metal chelates.

Some of the compounds described herein may also exist in theirtautomeric form.

The term “subject” includes both human and veterinary subjects.

A “therapeutically effective amount” or “diagnostically effectiveamount” refers to a quantity of a specified agent sufficient to achievea desired effect in a subject being treated with that agent. Ideally, atherapeutically effective amount or diagnostically effective amount ofan agent is an amount sufficient to inhibit or treat the disease withoutcausing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount or diagnostically effective amount ofan agent will be dependent on the subject being treated, the severity ofthe affliction, and the manner of administration of the therapeuticcomposition.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” is inclusive ofinhibiting the full development of a disease or condition, for example,in a subject who is at risk for a disease, or who has a disease, such ascancer or a disease associated with a compromised immune system.“Preventing” a disease or condition refers to prophylactic administeringa composition to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping a pathology or condition, or diminishing the severity of apathology or condition.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art. For a general discussion of prodrugs involving esters seeSvensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985).

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated.A variety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions which willnot affect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts, Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When anamine is deprotected, the resulting salt can readily be neutralized toyield the free amine Similarly, when an acid moiety, such as aphosphonic acid moiety is unveiled, the compound may be isolated as theacid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one ormore asymmetric centers; thus these compounds can exist in differentstereoisomeric forms. Accordingly, compounds and compositions may beprovided as individual pure enantiomers or as stereoisomeric mixtures,including racemic mixtures. In certain embodiments the compoundsdisclosed herein are synthesized in or are purified to be insubstantially enantiopure form, such as in a 90% enantiomeric excess, a95% enantiomeric excess, a 97% enantiomeric excess or even in greaterthan a 99% enantiomeric excess, such as in enantiopure form.

Groups which are substituted (e.g. substituted alkyl), may in someembodiments be substituted with a group which is substituted (e.g.substituted aryl). In some embodiments, the number of substituted groupslinked together is limited to two (e.g. substituted alkyl is substitutedwith substituted aryl, wherein the substituent present on the aryl isnot further substituted). In some embodiments, a substituted group isnot substituted with another substituted group (e.g. substituted alkylis substituted with unsubstituted aryl).

Overview

CRPC is responsible for all prostate cancer deaths, and eventually allprostate cancer will develop into CRPC. The current best treatment forCRPC is MDV3100 (enzalutamide), which binds to androgen receptor. It iseffective against a number of androgen-dependent prostate cancer celllines. However, it is ineffective against the androgen-dependentprostate cancer cell line 22Rv1. Compounds disclosed herein areeffective against all androgen-dependent cell lines tested including22Rv1, a promising and unique property.

Several of the compounds show sub-micromolar inhibition ofPSA-luciferase expression in C4-2 cells. Further, cell proliferation inandrogen-dependent cell lines is significantly decreased whileproliferation in androgen-independent cell lines is unaffected.

Agents

Disclosed herein are agents that can be used for treating prostatecancer, particularly castration-resistant prostate cancer. The agentsmay inhibit AR nuclear localization and/or reduce AR levels incastration-resistant prostate cancer.

In one embodiment, the agent is a compound, or a pharmaceuticallyacceptable salt or ester thereof, having a formula I of:

R²⁰—(Z)_(b)—(Y)_(c)—(R²¹)_(a)—(X)_(d)—R²²—R²³

wherein R²⁰ is an aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy,aryloxy, a thio-containing group, a seleno-containing group, halide, ora nitro-containing group;

Z is alkanediyl, substituted alkanediyl, cycloalkanediyl, or substitutedcycloalkanediyl;

Y is S, O, S(═O), —S(═O)(═O)—, or NR¹⁰, wherein R¹⁰ is H or alkyl(preferably methyl);

R²¹ is alkanediyl, substituted alkanediyl, cycloalkanediyl, substitutedcycloalkanediyl, alkadienyl, substituted alkadienyl, alkatrienyl, orsubstituted alkatrienyl;

X is —C(═O)—, —S(═O)(═O)—, or —N(H)C(═O)—;

R²² is a moiety that includes at least one divalent amino radical;

R²³ is an aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocycloalkyl, substituted heterocycloalkyl, alkoxy, aryloxy, amino,a thio-containing group, or a seleno-containing group;

a is 0 or 1;

b is 0 or 1;

c is 0 or 1; and

d is 0 or 1.

In some embodiments, if X is —C(═O)— then Y is not S. In certainembodiments, R²¹ is cycloalkanediyl, such as cyclopropanediyl. When R²¹is cycloalkanediyl, R²⁰ may be a phenyl optionally substituted with atleast one halogen and/or R²³ may be a phenyl substituted with at leastone halogen and or at least one alkyl.

In certain embodiments, R²⁰ is selected from isoxazolyl, substitutedisoxazolyl (e.g, dialkyl-substituted such as dimethyl,hydroxy-substituted, hydroxyalkyl-substituted, or a combinationthereof), oxazolyl, substituted oxazolyl (e.g, dialkyl-substituted suchas dimethyl, hydroxy-substituted, hydroxyalkyl-substituted, or acombination thereof) cyclohexyl, substituted cyclohexyl (e.g.,hydroxy-substituted cyclohexyl), piperidinyl, substituted piperidinyl(e.g., hydroxy-substituted piperidinyl), oxacyclopentyl, substitutedoxacyclopentyl (e.g., hydroxyalkyl-substituted), oxacyclohexanyl,substituted oxacyclopentyl (e.g., hydroxyalkyl-substituted), thiophenyl,substituted thiophenyl (e.g., hydroxyalkyl-substituted), phenyl,substituted phenyl (e.g., hydroxyalkyl-substituted orhalogen-substituted), pyridinyl, substituted pyridinyl (e.g.,hydroxyalkyl-substituted), indolyl, substituted indolyl (e.g.,hydroxyalkyl-substituted), furanyl, substituted furanyl (e.g.,hydroxyalkyl-substituted), imidazolyl, substituted imidazolyl (e.g.,hydroxyalkyl-substituted). In preferred embodiments, R²⁰ is substitutedisoxazolyl, particularly dialkyl (e.g., dimethyl)-substitutedisooxazolyl, phenyl, or substituted phenyl, particularlyhalogen-substituted phenyl (e.g., fluorophenyl).

In certain embodiments, R²¹ is selected from C₁-C₃ alkanediyl orsubstituted C₁-C₃ alkanediyl (e.g., alkyl-substituted such as methyl ordimethyl), preferably C₁ alkanediyl (—CH₂—), C₃ alkanediyl (—(CH₂)₃—),or cycloalkanediyl, preferably cyclopropanediyl. In certain embodiments,R²¹ is:

In certain embodiments, R²² is selected from:

wherein R¹¹ to R¹⁴ are each individually H or alkyl, provided that atleast one of R¹¹ to R¹⁴ is alkyl. In certain embodiments, R¹² and R¹³are each alkyl (e.g., methyl) and R¹¹ and R¹⁴ are each H. In certainembodiments, R¹¹ and R¹⁴ are each alkyl (e.g., methyl) and R¹² and R¹³are each H.

In certain embodiments, R²² is a divalent radical of a N-heterocyclicgroup. Illustrative N-heterocylic groups include piperazinyl,substituted piperazinyl, azabicyclo (for example, azabicyclooctane), andsubstituted azabicyclo.

In certain embodiments, R²³ is selected from phenyl, substituted phenyl(e.g., alkyl-substituted phenyl such as dimethyl-substituted, or halogensubstituted, such as chloro- or fluoro-substituted, oramino-substituted, or aminoalkyl-substituted; alkynyl-substitutedphenyl), piperidinyl, substituted piperidinyl (e g , amino-substituted),furanyl, substituted furanyl (e.g., aminoalkyl-substituted oramino-substituted), pyridinyl, substituted pyridinyl (e g ,aminoalkyl-substituted or amino-substituted), pyrimidinyl, substitutedpyrimidinyl (e g , aminoalkyl-substituted or amino-substituted),naphthenyl, substituted naphthenyl, (e g , aminoalkyl-substituted oramino-substituted), thiazole, substituted thiazole (e g ,aminoalkyl-substituted or amino-substituted); isoindazolyl, substitutedisoindazolyl (e g , aminoalkyl-substituted or amino-substituted);triazolyl, or substituted triazolyl (e g , aminoalkyl-substituted oramino-substituted). R²³ may have two or more substituents, such as analkyl substituent and a halogen substituent. Preferably, R²³ is asubstituted phenyl having a structure of:

wherein each of R¹-R⁵ is individually H, alkyl, substituted alkyl,alkynyl, substituted alkynyl, halogen, or cyano, provided that at leastone of R¹-R⁵ is not H. In certain embodiments, at least one of R¹-R⁵ isalkyl (such as methyl), halogen or cyano. In certain embodiments, R¹ isalkyl, halogen or cyano. In certain embodiments, R¹ is alkyl and R⁴ ishalogen. In certain embodiments, at least one of R¹-R⁵ ishydroxy-substituted alkynyl.

In certain embodiments, Z is selected from C₁-C₃ alkanediyl, preferably—CH₂—.

In certain embodiments, R²⁰ is phenyl or substituted isoxazolyl, b is 0;c is 1; a is 1; R²¹ is —CH₂—, Y is S; X is —S(═O)(═O)—, R²² is:

and R²³ is substituted phenyl.

In certain embodiments, R²⁰ is substituted phenyl, b is 0, c is 0, R²¹is cyclopropanediyl, a is 1, X is —C(═O)—, d is 1, R²² is

and R²³ is substituted phenyl. In one such embodiment, R²⁰ is halophenyland R²³ is halo- and alkyl-substituted phenyl.

In certain embodiments, R²¹ is —CH₂—, Y is S; and X is —S(═O)(═O)—.

In certain embodiments, R²² is:

In certain embodiments, Y is S, O, S(═O), —S(═O)(═O)—; and X is —C(═O)—.

In certain embodiments, b is 0; c is 0; a is 1; and X is —C(═O)—.

In certain embodiments, b is 0; c is 0; a is 1; X is —C(═O)—; and R²¹ isalkanediyl (particularly —CH₂CH₂—),

In certain embodiments, b is 0; c is 0; a is 1; X is —C(═O)—; R²¹ isalkanediyl (particularly —CH₂CH₂—),

and R²² is

In certain embodiments, b is 0; c is 0; a is 1; X is —C(═O)—; R²¹ isalkanediyl (particularly —CH₂CH₂—),

R²² is

R²⁰ is phenyl, substituted phenyl, or substituted isoxazolyl; and R²³ issubstituted phenyl.

In a further embodiment, the agent is a compound, or a pharmaceuticallyacceptable salt or ester thereof, having a formula II of:

R³⁰—(Z′)_(b)—(Y′)—(R³¹)_(a)—R³²—R³³

wherein R³⁰ is an aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, alkoxy,aryloxy, amino, a thio-containing group, or a seleno-containing group;

Z′ is alkanediyl, or substituted alkanediyl;

Y′ is S;

R³¹ is alkanediyl or substituted alkanediyl;

X is —C(═O)—;

R³² is a moiety that includes at least one divalent amino radical;

R³³ is a phenyl substituted with at least one halogen or cyano;

a is 0 or 1; and

b is 0 or 1.

In certain embodiments, R³⁰ is selected from isoxazolyl, substitutedisoxazolyl (e.g, dialkyl-substituted such as dimethyl,hydroxy-substituted, hydroxyalkyl-substituted, or a combinationthereof), oxazolyl, substituted oxazolyl (e.g, dialkyl-substituted suchas dimethyl, hydroxy-substituted, hydroxyalkyl-substituted, or acombination thereof) cyclohexyl, substituted cyclohexyl (e.g.,hydroxy-substituted cyclohexyl), piperidinyl, substituted piperidinyl(e.g., hydroxy-substituted piperidinyl), oxacyclopentyl, substitutedoxacyclopentyl (e.g., hydroxyalkyl-substituted), oxacyclohexanyl,substituted oxacyclopentyl (e.g., hydroxyalkyl-substituted), thiophenyl,substituted thiophenyl (e.g., hydroxyalkyl-substituted), phenyl,substituted phenyl (e.g., hydroxyalkyl-substituted), pyridinyl,substituted pyridinyl (e.g., hydroxyalkyl-substituted), indolyl,substituted indolyl (e.g., hydroxyalkyl-substituted), furanyl,substituted furanyl (e.g., hydroxyalkyl-substituted), imidazolyl,substituted imidazolyl (e.g., hydroxyalkyl-substituted). In preferredembodiments, R³⁰ is substituted isoxazolyl, particularly dialkyl (e.g.,dimethyl)-substituted isooxazolyl, or phenyl.

In certain embodiments, Z′ is selected from C₁-C₃ alkanediyl, preferably—CH₂—.

In certain embodiments, R³¹ is selected from C₁-C₃ alkanediyl orsubstituted C₁-C₃ alkanediyl (e.g., alkyl-substituted such as methyl ordimethyl), preferably C₁ alkanediyl.

In certain embodiments, R³² is selected from:

Preferably, R³³ is a substituted phenyl having a structure of:

wherein each of R¹-R⁵ is individually H, alkyl, halogen, or cyano,provided that at least one of R¹-R⁵ is halogen or cyano. In certainembodiments, R¹ is alkyl, halogen or cyano.

In certain embodiments, R³⁰ is substituted isoxazolyl, b is 1; a is 1;R²¹ is —CH₂—; and R³² is:

Certain embodiments are described below in the following numberedclauses:

1. A compound, or a pharmaceutically acceptable salt or ester thereof,selected from:

2. The compound of clause 1, wherein the compound is:

3. The compound of clause 1, wherein the compound is:

4. The compound of clause 1, wherein the compound is:

5. A pharmaceutical composition comprising at least one pharmaceuticallyacceptable additive, and a compound of any one of clauses 1-4.

6. The pharmaceutical composition of clause 5, wherein the compound is:

7. The pharmaceutical composition of clause 5, wherein the compound is:

8. The pharmaceutical composition of clause 5, wherein the compound is:

9. A method for treating prostate cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of acompound of any one of clauses 1-4.

10. The method of clause 9, wherein the prostate cancer iscastration-resistant prostate cancer.

11. The method of clause 9 or clause 10, wherein the compound is orallyadministered.

12. The method of any one of clauses 9-11, wherein the method is used incombination with androgen deprivation therapy.

13. The method of any one of clauses 9-12, wherein the agent isco-administered with abiraterone.

14. The method of any one of clauses 9-13, wherein the method furthercomprises identifying a subject that is in need of treatment with theagent.

15. The method of any one of clauses 9-14, wherein the compound is:

16. The method of any one of clauses 9-14, wherein the compound is:

17. The method of any one of clauses 9-14, wherein the compound is:

Illustrative compounds are shown in FIGS. 1A-1D.

FIG. 3 shows a synthesis of a parent structure that is amenable to themodifications lined out in a zone model. Isoxazole 2a can be obtainedfrom the chloromethylation of 3,5-dimethylisoxazole, or via thecorresponding alcohol, and can be converted to thiol 2b. In situalkylation of 2b with chloride 2d under the basic conditions of thiolateformation leads to 1. There are many methods known for pyridazinesynthesis, and the preparation of 2c can follow one of these methods,for example starting with the aniline Acylation of 2c with chloroacetylchloride provides 2d. FIG. 4 shows zones of modification for compound 1.The building blocks for zones 1 and 4 have been selected to cover alarge range of chemical diversity; in addition, they are commerciallyavailable and are therefore readily funneled into the segment-basedsynthesis plan. Zone 2 contains a few diamines that preserve thedistance between zone 1 and zone 3, i.e. where the nitrogens areappropriately spaced, but this zone can also be contracted to a simplenitrogen linker in order to probe the need to maintain the overalldistance and orientation between zone 1 and zone 4. Zone 3 containsanother spacer functionality, but the amide carbonyl group might also beinvolved in specific interactions with the binding site on the protein.Therefore, the distance between the carboxyl function and the halideelectrophile can be varied, and the carbonyl group can also be replacedby a sulfonyl function.

As described below, compounds 5a-h were synthesized directly fromcommercially available carboxylic acids 3a and N-arylated piperazines4a-h under amide coupling conditions with T3P (Scheme 2 (FIG. 5) andTable 1) (Basavaprabhhu et al., Synthesis 2013, 45, 1569-1601). Thediamine linker in zone 2 was examined in more detail through thesynthesis of analogs 5i-5m. For these target molecules, the requisitediamines 4i-m were prepared by a Buchwald-Hartwig cross-coupling of monoBoc-protected diamines with bromoarenes (Cabello-Sanchez et al., J. Org.Chem. 2007, 72, 2030-2039; Larsen et al., Tetrahedron 2008, 64,2938-2950). Reduction of amide 5b with lithium aluminum hydride led todiamine 6. For an initial set of zone 4 analogs, thioether 5b was alsooxidized to sulfoxide 12 and sulfone 13 in good yields with sodiumperiodate and m-chloroperbenzoate, respectively (Scheme 1, FIG. 5).

Additional zone 4 and zone 5 analogs with a phenyl group in place of theisoxazole ring were obtained from carboxylic acids 3b-3g (Scheme 2 (FIG.6) and Table 1). Coupling to piperazine 4b provided amides 7-11 and 16in high yields. Alkynyl amide 10 was further hydrogenated to cis-alkene14 using a Lindlar catalyst. The cis-cyclopropane 15 was prepared by aSimmons-Smith cyclopropanation of cis-alkene 14 (Concellón et al., Org.Lett. 2007, 9, 2981-2984), whereas the trans-cyclopropane 16 wasobtained by coupling of commercially availabletrans-2-phenylcyclopropanecarboxylic acid 3g with piperazine 4b.

Further modifications in zones 3-4 were accomplished by acylation ofpiperazine 4b with either 2-chloroacetyl chloride orchloromethanesulfonyl chloride to form the corresponding amide 17a orsulfonamide 17b in good yields (Scheme 3 (FIG. 7) and Table 1).S_(N)2-reaction of 17a and 17b led to ether 18a, amine 18b, andthioether 18c. Starting with carboxylic acid 3a, urea 20a and carbamate20b were obtained in moderate yields via a Curtius rearrangement andaddition of the intermediate isocyanate 19 to amine 4b and alcohol 4n,respectively (Scheme 4, FIG. 7) (WO 2005/085275).

A bridged bicyclic ring was introduced to add a strong conformationalconstraint in zone 2 (Scheme 5 (FIG. 8) and Table 1). Boc-protection ofnortropinone hydrochloride 21 followed by enolization with NaHMDS andtrapping of the enolate with N-phenyltriflimide provided vinyl triflate22 in good yield. A Suzuki coupling was used to install the o-tolylgroup, and the styrene double bond was reduced with Pd/C to afford 23 asa mixture of diastereomers. Without separation, this mixture wasdeprotected and acylated with α-chloroacetyl chloride. Finally, thechloride was displaced using thiol 25 and sodium hydride to afford thethioether. Diastereomers 26a and 26b were separated by chromatography onSi02 to afford both analogs in modest yields.

TABLE 1 Structures of amine building blocks 4 and analogs 5, 7-11, and16. Analog Amine 4 R X 5a

Ph — 5b

(2-Me)Ph — 5c

(3-Me)Ph — 5d

(4-Me)Ph — 5e

(2-NC)Ph — 5f

(2-F)Ph — 5g

1-Naphthyl — 5h

(2-MeO)Ph — 5i

(2-Me)Ph — 5j

(2-Me)Ph — 5k

(2-Me)Ph — 5l

Ph — 5m

(3-Me)Ph —  7

(2-Me)Ph CH₂SCH₂  8

(2-Me)Ph (CH₂)₃  9

(2-Me)Ph SCH₂ 10

(2-Me)Ph C≡C 11

(2-Me)Ph (E)-HC═CH 16

(2-Me)Ph (E)-c-C₃H₄

Pharmaceutical Compositions and Method of Use

The agents disclosed herein may be administered to a subject fortreating prostate cancer, particularly castration-resistant prostatecancer. In certain embodiments a subject is identified as havingcastration-resistant prostate cancer that may be responsive to theagents disclosed herein. For example, patients that are offered any formof androgen deprivation therapy or anti-androgen therapy, includingtreatment with abiraterone or MDV3100, for the management of prostatecancer would be candidates for treatment with the agents disclosedherein.

Administration of the agent may reduce the nuclear level of androgenreceptor in castration-resistant prostate cancer (CRPC) cells relativeto the untreated control CRPC cells. Reducing nuclear androgen receptorlevels is expected to inhibit its activation. Reduction of androgenreceptor activation can be determined via measuring androgen-responsivegenes, such as prostate-specific antigen (PSA).

In certain embodiments, the agent may be co-administered with anothertherapeutic agent such as, for example, an immunostimulant, ananti-cancer agent, an antibiotic, or a combination thereof. Inparticular, the agents targeting AR nuclear localization could be usedin combination with standard androgen deprivation therapy (ADT) or withabiraterone in the treatment of CRPC. In one embodiment, the agent isco-administered with MDV3100 (enzalutamide), which may producesynergistic results since MDV3100 targets the ligand binding domainwhereas the agent targets other domain(s) of the androgen receptor.

The agents disclosed herein can be included in a pharmaceuticalcomposition for administration to a subject. The pharmaceuticalcompositions for administration to a subject can include at least onefurther pharmaceutically acceptable additive such as carriers,thickeners, diluents, buffers, preservatives, surface active agents andthe like in addition to the molecule of choice. Pharmaceuticalcompositions can also include one or more additional active ingredientssuch as antimicrobial agents, anti-inflammatory agents, anesthetics, andthe like. The pharmaceutically acceptable carriers useful for theseformulations are conventional. Remington's Pharmaceutical Sciences, byE. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995),describes compositions and formulations suitable for pharmaceuticaldelivery of the compounds herein disclosed.

The pharmaceutical compositions may be in a dosage unit form such as aninjectable fluid, an oral delivery fluid (e.g., a solution orsuspension), a nasal delivery fluid (e.g., for delivery as an aerosol orvapor), a semisolid form (e.g., a topical cream), or a solid form suchas powder, pill, tablet, or capsule forms.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

The agents disclosed herein can be administered to subjects by a varietyof mucosal administration modes, including by oral, rectal, intranasal,intrapulmonary, or transdermal delivery, or by topical delivery to othersurfaces. Optionally, the agents can be administered by non-mucosalroutes, including by intramuscular, subcutaneous, intravenous,intra-arterial, intra-articular, intraperitoneal, intrathecal,intracerebroventricular, or parenteral routes. In other alternativeembodiments, the agents can be administered ex vivo by direct exposureto cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the agents can be combinedwith various pharmaceutically acceptable additives, as well as a base orvehicle for dispersion of the compound. Desired additives include, butare not limited to, pH control agents, such as arginine, sodiumhydroxide, glycine, hydrochloric acid, citric acid, and the like. Inaddition, local anesthetics (for example, benzyl alcohol), isotonizingagents (for example, sodium chloride, mannitol, sorbitol), adsorptioninhibitors (for example, Tween 80 or Miglyol 812), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included. Adjuvants, such as aluminum hydroxide (forexample, Amphogel, Wyeth Laboratories, Madison, N.J.), Freund'sadjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many othersuitable adjuvants well known in the art, can be included in thecompositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The agents can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The agents can be combined with the base or vehicle according to avariety of methods, and release of the agents can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the agent is dispersed in microcapsules(microspheres) or nanocapsules (nanospheres) prepared from a suitablepolymer, for example, isobutyl 2-cyanoacrylate (see, for example,Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in abiocompatible dispersing medium, which yields sustained delivery andbiological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the agents can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compound can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the agents can be administered in a time releaseformulation, for example in a composition which includes a slow releasepolymer. These compositions can be prepared with vehicles that willprotect against rapid release, for example a controlled release vehiclesuch as a polymer, microencapsulated delivery system or bioadhesive gel.Prolonged delivery in various compositions of the disclosure can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monostearate hydrogels and gelatin.When controlled release formulations are desired, controlled releasebinders suitable for use in accordance with the disclosure include anybiocompatible controlled release material which is inert to the activeagent and which is capable of incorporating the compound and/or otherbiologically active agent. Numerous such materials are known in the art.Useful controlled-release binders are materials that are metabolizedslowly under physiological conditions following their delivery (forexample, at a mucosal surface, or in the presence of bodily fluids).Appropriate binders include, but are not limited to, biocompatiblepolymers and copolymers well known in the art for use in sustainedrelease formulations. Such biocompatible compounds are non-toxic andinert to surrounding tissues, and do not trigger significant adverseside effects, such as nasal irritation, immune response, inflammation,or the like. They are metabolized into metabolic products that are alsobiocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon.-caprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, theagent can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the agent is administered to a subject in need of suchtreatment for a time and under conditions sufficient to prevent,inhibit, and/or ameliorate a selected disease or condition or one ormore symptom(s) thereof.

The administration of the agent can be for either prophylactic ortherapeutic purpose. When provided prophylactically, the agent isprovided in advance of any symptom. The prophylactic administration ofthe agents serves to prevent or ameliorate any subsequent diseaseprocess. When provided therapeutically, the compound is provided at (orshortly after) the onset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the agent can be administeredto the subject by the oral route or in a single bolus delivery, viacontinuous delivery (for example, continuous transdermal, mucosal orintravenous delivery) over an extended time period, or in a repeatedadministration protocol (for example, by an hourly, daily or weekly,repeated administration protocol). The therapeutically effective dosageof the agent can be provided as repeated doses within a prolongedprophylaxis or treatment regimen that will yield clinically significantresults to alleviate one or more symptoms or detectable conditionsassociated with a targeted disease or condition as set forth herein.Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat, avian,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models. Using such models, only ordinarycalculations and adjustments are required to determine an appropriateconcentration and dose to administer a therapeutically effective amountof the compound (for example, amounts that are effective to elicit adesired immune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the agents may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the agents will vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the agent for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the agent is outweighed in clinical terms bytherapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of an agent within the methods andformulations of the disclosure is about 0.01 mg/kg body weight to about20 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg bodyweight, or about 0.2 mg/kg to about 2 mg/kg body weight. Dosage can bevaried by the attending clinician to maintain a desired concentration ata target site (for example, the lungs or systemic circulation). Higheror lower concentrations can be selected based on the mode of delivery,for example, trans-epidermal, rectal, oral, pulmonary, or intranasaldelivery versus intravenous or subcutaneous delivery. Dosage can also beadjusted based on the release rate of the administered formulation, forexample, of an intrapulmonary spray versus powder, sustained releaseoral versus injected particulate or transdermal delivery formulations,and so forth.

EXAMPLES 1. Biological Materials and Methods Materials

Phosphate buffered saline (PBS) solution was purchased from FisherScientific (MA, USA). Trypsin-EDTA solution, dimethyl sulfoxide (DMSO),Roswell Park Memorial Institute (RPMI) 1640 medium, ethanol (200 proof),puromycin powder, and G418 powder were purchased from Sigma-Aldrich (MO,USA). Fetal bovine Serum (FBS), penicillin-streptomycin solution werepurchased from Invitrogen (NY, USA). Dual-Luciferase® Reporter AssaySystem was purchased from Promega (WI, USA). PSA6.1-luc plasmid was agift from Dr. Marianne Sadar at the University of British Columbia (BC,CA) and pRL-TK Renilla luciferase reporter plasmid was purchased fromPromega (WI, USA). The C4-2 castration-resistant prostate cancer cellline was kindly provided by Dr. Leland W. K. Chung (Cedars-Sinai MedicalCenter).

2. Chemistry General

Moisture and air-sensitive reactions were performed under N₂ or Aratmosphere and glassware used for these reactions was flamed dried andcooled under N₂ or Ar prior to use. THF and Et₂O were distilled fromsodium/benzophenone ketyl. DMF and CH₂Cl₂ were distilled from CaH₂.1,4-Dioxane was purchased from Acros (Sure/Seal bottle) and used asreceived. Et₃N was distilled from CaH₂ and stored over KOH. Toluene waspurified by passage through an activated alumina filtration system.Melting points were determined using a Mel-Temp II instrument and arenot corrected. Infrared spectra were determined using a Smiths DetectionIdentifyIR FT-IR spectrometer. High-resolution mass spectra wereobtained on a Micromass UK Limited, Q-TOF Ultima API, Thermo ScientificExactive Orbitrap LC-MS. Automated column chromatography was done usingan Isco Combiflash Rf. ¹H and ¹³C NMR spectra were obtained on BrukerAdvance 300 MH₂, 400 MHz, or 500 MH₂ instruments. Chemical shifts (δ)were reported in parts per million with the residual solvent peak usedas an internal standard, δ ¹H/¹³C (Solvent): 7.26/77.00 (CDCl₃);2.05/29.84 (acetone-d6); 2.50/39.52 (DMSO-d6), 3.31/49.00 (CD3OD); andare tabulated as follows: chemical shift, multiplicity (s=singlet,brs=broad singlet, d=doublet, brd=broad doublet, t=triplet, appt=apparent triplet, q=quartet, m=multiplet), number of protons, andcoupling constant(s). ¹³C NMR spectra were obtained at 75 MH₂, 100 MHz,or 125 MH₂ using a proton-decoupled pulse sequence and are tabulated byobserved peak. CDCl₃ was filtered through dried basic alumina prior touse. Thin-layer chromatography was performed using pre-coated silica gel60 F₂₅₄ plates (EMD, 250 μm thickness) and visualization wasaccomplished with a 254 nm UV light and by staining with a PMA solution(5 g of phosphomolybdic acid in 100 mL of 95% EtOH), Vaughn's reagent(4.8 g of (NH4)₆Mo₇O₂₄.4H₂O and 0.2 g of Ce(SO₄)₂ in 100 mL of a 3.5 NH₂SO₄ solution) or a KMnO₄ solution (1.5 g of KMnO₄ and 1.5 g of K₂CO₃in 100 mL of a 0.1% NaOH solution). Chromatography on SiO₂ (Silicycle,Silia-P Flash Silica Gel or SiliaFlash® P60, 40-63 μm) was used topurify crude reaction mixtures. Final products were >95% purity asanalyzed by RP (reverse phase) HPLC (Alltech Prevail C-18, 100×4.6 mm, 1mL/min, CH₃CN, H₂O and 0.1% TFA) with UV (210, 220 and 254 nm), ELS(nebulizer 45° C., evaporator 45° C., N₂ flow 1.25 SLM), and MSdetection using a Thermo Scientific Exactive Orbitrap LC-MS (ESIpositive). All other materials were obtained from commercial sources andused as received.

Example 1 Synthesis and Characterization

Synthesis of several of the compounds is described in detail below:

-   tert-Butyl 4-(3-bromo-2-methylphenyl)piperazine-1-carboxylate    (BRE454-64). A microwave vial under Ar was charged with tert-butyl    1-piperazinecarboxylate (154 mg, 0.825 mmol), NaO-t-Bu (0.0952 g,    0.990 mmol), (rac)-BINAP (0.0393 g, 0.0619 mmol, 7.5 mol %),    Pd₂(dba)₃ (0.0192 g, 0.0206 mmol), and degassed toluene (2.1 mL).    2-Bromo-6-iodotoluene (121 μL, 0.825 mmol) was added, and the    mixture was heated in sealed vial at 80° C. for 19 h, cooled to room    temperature, diluted with CH₂Cl₂, filtered through Celite, and    concentrated in vacuo. The mixture was purified by chromatography on    SiO₂ (1:9, EtOAc/hexanes) to give the product (0.095 g, 0.27 mmol,    32%) as a yellow oil: ¹H NMR (500 MHz, CDCl₃) δ 7.30 (d, J=8.0 Hz, 1    H), 7.02 (t, J=8.0 Hz, 1 H), 6.95 (d, J=7.5 Hz, 1 H), 3.57 (m, 4 H),    2.83 (t, J=4.5 Hz, 4 H), 2.40 (s, 3 H), 1.49 (s, 9 H).

-   1-(4-(3-Bromo-2-methylphenyl)piperazin-1-yl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)    ethan-1-one (BRE454-75). A solution of BRE454-64 (0.0770 g, 0.22    mmol) in THF (0.3 mL) at 0° C. was treated with 4 M HCl in dioxane    (1.3 mL) and stirred at 0° C. for 2 h. The yellow solid was    collected by filtration, washed with Et₂O, dried under high vacuum    and carried on to the next step without further purification.-   To a solution of ([(3,5-dimethylisoxazol-4-yl)methyl]thio)acetic    acid (0.0350 g, 0.174 mmol) in CH₂Cl₂ (1.7 mL) was added    4-(3-bromo-2-methylphenyl)piperazine hydrochloride and triethylamine    (121 μL, 0.870 mmol). The mixture was cooled to 0° C., treated with    T3P (50% solution in EtOAc, 184 μL, 0.261 mmol), warmed to room    temperature, stirred for 20 h, diluted with CH₂Cl₂, and washed with    sat. NH₄Cl, sat. NaHCO₃, and brine. The organic layer was dried    (Na₂SO₄), filtered, and concentrated in vacuo. The crude material    was purified by chromatography on SiO₂ (3:2, EtOAc/hexanes, base    washed with 0.1% Et₃N prior to use) to give the product (0.0762 g,    0.174 mmol, quant. 100% pure by ELSD) as a colorless oil: IR (ATR)    2921, 2820, 1637, 1587, 1562, 1460, 1428, 1282, 1237, 1195, 1136,    1038, 994, 913, 780, 731, 714 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.32    (dd, J=0.8, 7.6 Hz, 1 H), 7.03 (t, J=8.0 Hz, 1 H), 6.94 (dd, J=0.8,    8.0 Hz, 1 H), 3.77 (br s, 2 H), 3.63 (s, 2 H), 3.63-3.57 (m, 2 H),    3.23 (s, 2 H), 2.90 (t, J=4.4 Hz, 2 H), 2.88-2.83 (m, 2 H), 2.43 (s,    3 H), 2.40 (s, 3 H), 2.31 (s, 3 H); ¹³C-NMR (125 MHz, CDCl₃) δ    167.6, 166.8, 159.7, 152.2, 132.9, 128.1, 127.4, 126.6, 118.3,    109.7, 52.1, 51.8, 46.8, 42.2, 32.1, 23.8, 18.2, 11.1, 10.2; HRMS    (ESI) m/z calcd for C₁₉H₂₅N₃O₂BrS ([M+H]⁺) 438.0845, found 438.0831.

-   tert-Butyl 4-(o-tolyl)-1,4-diazepane-1-carboxylate (BRE454-66). A    microwave vial under Ar was charged with 1-Boc-homopiperazine (223    mg, 1.10 mmol), NaO-t-Bu (0.116 g, 1.20 mmol), (rac)-BINAP (0.0478    g, 0.0752 mmol, 7.5% mol), Pd₂(dba)₃ (0.0233 g, 0.0251 mmol, 2.5%    mol in Pd), and degassed toluene (2.8 mL). 2-Bromotoluene (0.175 g,    1.00 mmol) was added, and the mixture was heated in a sealed vial at    80° C. for 19 h, cooled to room temperature diluted with CH₂Cl₂,    filtered over Celite, and concentrated. The crude material was    purified by chromatography on SiO₂ (1:9, EtOAc/hexanes) to give the    product (0.139 g, 0.479 mmol, 48%) as a yellow oil: IR (ATR) 2973,    2828, 1689, 1598, 1491, 1457, 1411, 1364, 1233, 1215, 1156, 1122,    878, 761, 725 cm⁻¹; ¹H NMR (500 MHz, CDCl₃, rt, rotamers) δ 7.16 (d,    J=6.0 Hz, 1 H), 7.12 (d, J=6.0 Hz, 1 H), 7.04 (d, J=7.5 Hz, 1 H),    6.95 (t, J=7.0 Hz, 1 H), 3.62-3.52 (m, 4 H), 3.12-3.04 (m, 4 H),    2.31 (s, 3 H), 2.00-1.88 (m, 2 H), 1.49 (s, 9 H); ¹³C-NMR (100 MHz,    CDCl₃, rt, rotamers) 6155.6, 155.5, 153.9, 153.8, 132.9, 130.9,    126.5, 123.1, 120.8 (2 C), 79.3, 56.2, 56.0, 55.5, 55.2, 48.4, 48.0,    46.2, 45.4, 29.0, 28.9, 28.5, 18.5; HRMS (ESI) m/z calcd for    C₁₇H₂₇N₂O₂ ([M+H]⁺) 291.2067, found 291.2062.

-   1-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-2-4(3,5-dimethylisoxazol-4-yl)methyl)thio)    ethan-1-one (BRE454-58). To a solution of    ([(3,5-dimethylisoxazol-4-yl)methyl]thio)acetic acid (0.0450 g,    0.224 mmol) in CH₂Cl₂ (2.2 mL) was added    1-(5-chloro-2-methylphenyl)piperazine (0.0565 g, 0.268 mmol) and    triethylamine (93 μL, 0.671 mmol). The mixture was cooled to 0° C.,    treated with T3P (50% solution in EtOAc, 237 μL, 0.335 mmol), warmed    to room temperature, stirred for 20 h, diluted with CH₂Cl₂, and    washed with sat. NH₄Cl, sat. NaHCO₃, and brine. The organic layer    was dried (Na₂SO₄), filtered, and concentrated in vacuo. The crude    material was purified by chromatography on SiO₂ (1:1, EtOAc/hexanes,    base washed with 0.1% Et₃N prior to use) to give the product (0.0881    g, 0.224 mmol, quant, 99.9% pure by ELSD) as a clear colorless oil:    IR (ATR) 2921, 2818, 1635, 1592, 1489, 1438, 1270, 1224, 1195, 1148,    1039, 924, 910, 818, 728 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.11 (d,    J=8.0 Hz, 1 H), 6.99 (dd, J=2.0, 8.0 Hz, 1 H), 6.94 (d, J=2.4 Hz, 1    H), 3.76 (t, J=4.8 Hz, 2 H), 3.63 (s, 2 H), 3.59 (t, J=4.8 Hz, 2 H),    3.23 (s, 2 H), 2.91 (t, J=4.8 Hz, 2 H), 2.86 (t, J=4.8 Hz, 2 H),    2.43 (s, 3 H), 2.30 (s, 3 H), 2.27 (s, 3 H); ¹³C NMR (125 MHz,    CDCl₃) δ 167.6, 166.8, 159.7, 151.7, 132.1, 131.8, 130.9, 123.7,    119.7, 109.7, 51.6, 51.5, 46.8, 42.2, 32.0, 23.7, 17.4, 11.1, 10.2;    HRMS (ESI) m/z calcd for C₁₉H₂₅N₃O₂ClS ([M+H]⁺) 394.1351, found    394.1340.

-   1-(((Phenylthio)methyl)sulfonyl)-4-(o-tolyl)piperazine (BRE454-84).    A solution of 1-(2-methylphenyl)piperazine (0.500 g, 2.75 mmol) and    triethylamine (0.39 mL, 2.75 mmol) in CH₂Cl₂ (9.8 mL) at 0° C. was    treated with chloromethanesulfonyl chloride (0.460 g, 3.03 mmol),    gradually warmed to room temperature, and stirred for 14 h. The    reaction mixture was quenched with sat. NH₄Cl (3 mL) and extracted    with EtOAc (3×20 mL). The combined organic portion was washed with    water (2×10 mL) and brine (10 mL), dried (Na₂SO₄), filtered, and    concentrated. The crude solid was filtered through a plug of SiO₂    (pretreated with 0.1% Et₃N in 30% EtOAc/hexanes) and washed    thoroughly with 30% EtOAc/hexanes to give the product as an orange    solid (0.676 g, 2.34 mmol, 85%): NMR (400 MHz, CDCl₃) δ 7.21-7.17    (m, 2 H), 7.05-7.00 (m, 2 H), 4.57 (s, 2 H), 3.63 (t, J=4.8 Hz, 4    H), 2.98 (t, J=5.2 Hz, 4 H), 2.31 (s, 3 H).-   A solution of this product (0.0400 g, 0.139 mmol), thiophenol    (0.0610 g, 0.554 mmol), and Cs₂CO₃ (0.0903 g, 0.277 mmol) in DMF    (0.28 mL) was stirred at 80° C. for 2 d. The reaction mixture was    diluted with brine (10 mL) and extracted with EtOAc (20 mL). The    organic layer was separated, washed with brine (2×10 mL), dried    (Na₂SO₄), and concentrated in vacuo. The crude material was purified    by chromatography on SiO₂ (1:4, EtOAc/hexanes) the product as a    clear colorless oil (0.0257 g, 0.0709 mmol, 51%): IR (ATR) 3054,    2918, 2823, 1598, 1581, 1493, 1440, 1342, 1324, 1262, 1225, 1153,    1112, 1070, 954, 765, 744, 725, 691 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ    7.59 (d, J=7.5 Hz, 2 H), 7.39-7.30 (m, 3 H), 7.21-7.14 (m, 2 H),    7.02 (t, J=7.5 Hz, 1 H), 6.98 (d, J=8.0 Hz, 1 H), 4.33 (s, 2 H),    3.51 (t, J=4.5 Hz, 4 H), 2.92 (t, J=4.5 Hz, 4 H), 2.28 (s, 3 H); ¹³C    NMR (100 MHz, CDCl₃) δ 150.7, 133.4, 132.7, 131.2, 131.1, 129.4,    128.1, 126.7, 123.9, 119.4, 54.2, 51.8, 46.8, 17.7; HRMS (+ESI) m/z    calcd for C₁₈H₂₃N₂O₂S2 ([M+H]⁺) 363.1195, found 363.1190.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-phenylpiperazin-1-yl)ethanone    (5a). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid 3a (0.0200 g,    0.0994 mmol) in CH₂Cl₂ (1.25 mL) was added 1-phenylpiperazine 4a    (0.0190 g, 0.119 mmol) and Et₃N (41 μL, 0.298 mmol). The reaction    mixture was cooled to 0° C., treated with T3P (50 wt. % solution in    EtOAc, 105 μL, 0.149 mmol), allowed to warm to room temperature,    stirred for 2 d, diluted with CH₂Cl₂ and washed with satd. aqueous    NH₄Cl, satd. aqueous NaHCO₃, brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-100%), product eluted at 60%) to give 5a    (0.0330 g, 0.0955 mmol, 96%, 100% pure by ELSD) as a colorless    solid: Mp 74-75° C.; IR (ATR) 2856, 2802, 1627, 1599, 1496, 1440,    1416, 1229, 1141, 1034, 909, 765, 698 cm⁻¹; ¹HNMR (300 MHz, CDCl₃) δ    7.26-7.21 (m, 1 H), 6.89-6.83 (m, 3 H), 3.72 (app t, 2 H, J=5.2 Hz),    3.56 (s, 2 H), 3.56-3.54 (m, 2 H), 3.18 (s, 2 H), 3.15-3.10 (m, 2    H), 2.34 (s, 3 H), 2.23 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 166.5,    165.8, 158.6, 149.8, 128.2, 119.6, 115.6, 108.7, 48.5, 48.3, 45.3,    40.7, 31.0, 22.7, 10.0, 9.1; HRMS (ESI) m/z calcd for C₁₈H₂₄N₃O₂S    ([M+H]⁺) 346.1584, found: 346.1571.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(o-tolyl)piperazin-1-yl)ethanone    (5b). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0200    g, 0.0994 mmol) in CH₂Cl₂ (1.25 mL) was added 1-(o-tolyl)piperazine    4b (0.0210 g, 0.119 mmol) and Et₃N (41 μL, 0.298 mmol). The reaction    mixture was cooled to 0° C., treated with T3P (50 wt. % solution in    EtOAc, 105 μL, 0.149 mmol), allowed to warm to room temperature,    stirred for 2 d, diluted with CH₂Cl₂ and washed with satd. aqueous    NH₄Cl, satd. aqueous NaHCO₃, brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-100%), product eluted at 40%) to give 5b    (0.0348 g, 0.0968 mmol, 97%, 100% pure by ELSD) as a colorless    solid: Mp 89-91° C.; IR (ATR) 2959, 2828, 1631, 1492, 1430, 1261,    1226, 1138, 1036, 979, 959, 776, 726 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ    7.18 (dd, 2 H, J=9.0, 7.5 Hz), 7.01 (dd, 2 H, J=14.1, 9.0 Hz), 3.76    (app t, 2 H, J=4.9 Hz), 3.63 (s, 2 H), 3.59 (app t, 2 H, J=4.9 Hz),    3.24 (s, 2 H), 2.93 (app t, 2 H, J=4.9 Hz), 2.88 (app t, 2 H, J=4.9    Hz), 2.43 (s, 3 H), 2.32 (s, 3 H), 2.30 (s, 3 H); ¹³C NMR (75 MHz,    CDCl₃) δ 166.5, 165.8, 158.7, 149.6, 131.7, 130.2, 125.7, 122.8,    118.1, 108.7, 50.8, 50.6, 46.0, 41.3, 31.1, 22.7, 16.7, 10.0, 9.1;    HRMS (ESI) m/z calcd for C₁₉H₂₆N₃O₂S ([M+H]⁺) 360.1740, found    360.1725.

2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(m-tolyl)piperazin-1-yl)ethanone(5c). A solution of 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)aceticacid (3a, 0.0200 g, 0.0994 mmol) in CH₂Cl₂ (1.25 mL) was added1-(m-tolyl)piperazine (4c, 21 μL, 0.119 mmol), Et₃N (41 μL, 0.298 mmol).The reaction mixture was cooled to 0° C., treated with T3P (50 wt. %solution in EtOAc, 105 μL, 0.149 mmol), allowed to warm to roomtemperature, stirred for 2 d, diluted with CH₂Cl₂ and washed with satd.aqueous NH₄Cl, satd. aqueous NaHCO₃, brine, dried (Na₂SO₄), filtered,and concentrated in vacuo. The crude residue was purified bychromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,EtOAc/hexanes gradient (10-100%), eluted at 60%) to give 5c (0.0343 g,0.954 mmol, 96%, 99.5% pure by ELSD) as a yellow oil: IR (ATR) 2918,2819, 1635, 1600, 1493, 1424, 1244, 1192, 1145, 995, 957, 775, 729, 694cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.17 (app t, 1 H, J=7.8 Hz), 6.75-6.72(m, 3 H), 3.76 (app t, 2 H, J=5.2 Hz), 3.61 (s, 2 H), 3.60-3.58 (m, 2H), 3.23 (s, 2 H), 3.17 (ddd, 4 H, J=5.5, 5.2, 5.0 Hz), 2.41 (s, 3 H),2.32 (s, 3 H), 2.28 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 166.5, 165.8,158.6, 149.8, 138.0, 128.1, 120.5, 116.5, 112.8, 108.7, 48.6, 48.5,45.3, 40.8, 31.0, 22.7, 20.7, 10.0, 9.1; HRMS (ESI) m/z calcd forC₁₉H₂₆N₃O₂S ([M+H]⁺) 360.1740, found: 360.1725.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(p-tolyl)piperazin-1-yl)ethanone    (5d). A solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0200    g, 0.0994 mmol) in CH₂Cl₂ (1.25 mL) was added 1-(p-tolyl)piperazine    (4d, 21 μL, 0.119 mmol), Et₃N (41 μL, 0.298 mmol). The reaction    mixture was cooled to 0° C., treated with T3P (50 wt. % solution in    EtOAc, 105 μL, 0.149 mmol), allowed to warm to room temperature,    stirred for 2 d, diluted with CH₂Cl₂, washed with satd. aqueous    NH₄Cl, satd. aqueous NaHCO₃, and brine, dried (Na₂SO₄), filtered,    and concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient ((10-100%), eluted at 60%) to give 5d (0.0266    g, 0.0740 mmol, 74%, 100% pure by ELSD) as a red solid: Mp 83-85°    C.; IR (ATR) 2855, 2801, 1627, 1514, 1440, 1416, 1261, 1230, 1142,    1043, 960, 815, 724 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.10 (d, 2 H,    J=8.1 Hz), 6.85 (d, 2 H, J=8.1 Hz), 3.77 (app t, 2 H, J=4.7 Hz),    3.61-3.58 (m, 4 H), 3.23 (s, 2 H), 3.13 (ddd, 4 H, J=5.6, 5.5, 4.7    Hz), 2.41 (s, 3 H), 2.28, (s, 6 H); ¹³C NMR (75 MHz, CDCl₃) δ 167.5,    166.8, 159.7, 148.7, 130.3, 129.8, 117.0, 109.7, 50.1, 49.9, 46.4,    41.8, 32.1, 23.7, 20.4, 11.0, 10.1; HRMS (ESI) m/z calcd for    C₁₉H₂₆N₃O₂S ([M+H]⁺) 360.1740, found 360.1725.

-   2-(4-(2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)acetyppiperazin-1-yl)benzonitrile    (MK415-62; 5e). To a solution of    ([(3,5-dimethylisoxazol-4-yl)methyl]thio)acetic acid (3a, 0.0280 g,    0.132 mmol) in CH₂Cl₂ (1.3 mL) was added    2-(piperazin-1-yl)benzonitrile (4e, 0.0253 g, 0.132 mmol) and Et₃N    (56 μL, 0.400 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 140 μL, 0.200 mmol),    allowed to warm to room temperature, stirred for 20 h, diluted with    CH₂Cl₂, washed with satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and    brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The    crude residue was purified by chromatography on SiO₂ (95:5,    CH₂Cl₂/MeOH) to give 5e (0.0390 g, 0.105 mmol, 80%, 99.9% pure by    ELSD) as a yellow solid: Mp 142-143° C.; IR (neat) 2919, 2216, 1637,    1593, 1420, 1232 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 7.61 (dd, 1 H,    J=7.6, 1.6 Hz), 7.51 (ddd, 1 H, J=8.4, 7.6, 1.6 Hz), 7.09 (dt, 1 H,    J=7.6, 0.9 Hz), 7.02 (d, 1 H, J=8.4 Hz), 3.82 (app t, 2 H, J=4.8    Hz), 3.67 (app t, 2 H, J=4.8 Hz), 3.62 (s, 2 H), 3.24 (s, 2 H),    3.24-3.21 (m, 2 H) 3.15 (app t, 2 H, J=5.4 Hz), 2.41 (s, 3 H), 2.28    (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.6, 166.7, 159.6, 154.9,    134.3, 133.9, 122.7, 118.9, 118.0, 109.7, 106.7, 51.9, 51.1, 46.6,    41.8, 32.1, 23.7, 11.0, 10.1; HRMS (ESI) m/z calcd for C₁₉H₂₃N₄O₂S    ([M+H]⁺) 371.1542, found 371.1536.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2-fluorophenyl)piperazin-1-yl)ethan-1-one    (BRE454-54; 5f). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0758    g, 0.377 mmol) in CH₂Cl₂ (3.8 mL) was added    1-(2-fluorophenyl)-piperazine (4f, 0.0814 g, 0.452 mmol) and Et₃N    (262 μL, 1.88 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 399 μL, 0.565 mmol),    allowed to warm to room temperature, stirred for 20 h, diluted with    CH₂Cl₂, and washed with satd. aqueous NH₄Cl solution, satd. aqueous    NaHCO₃ solution, and brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (3:2, EtOAc/hexanes, base washed with 0.1%    Et₃N prior to use) to give 5f (0.134 g, 0.369 mmol, 98%, 100% pure    by ELSD) as a light yellow oil: IR (ATR) 2918, 2827, 1636, 1613,    1500, 1439, 1237, 1195, 1147, 1031, 909, 811, 753, 725 cm⁻¹; ¹H NMR    (400 MHz, CDCl₃) δ 7.10-6.90 (m, 4 H), 3.79 (app t, 2 H, J=5.2 Hz),    3.63-3.59 (m, 4 H), 3.23 (s, 2 H), 3.10 (app t, 2 H, J=4.8 Hz), 3.05    (app t, 2 H, J=5.2 Hz), 2.28 (s, 3 H), 2.42 (s, 3 H); ¹³C NMR (125    MHz, CDCl₃) δ 167.5, 166.8, 159.7, 155.7 (d, J_(C—F)=245.0 Hz),    139.4 (d, J_(C—F)=8.8 Hz), 124.5 (d, J_(C—F)=3.8 Hz), 123.3 (d,    J_(C—F)=8.8 Hz), 119.2 (d, f_(c) F=2.5 Hz), 116.3 (d, J_(C—F)=20.0    Hz), 109.7, 50.7 (d, J_(C—F)=2.5 Hz), 50.3 (d, J_(C—F)=2.5 Hz),    46.6, 41.9, 32.1, 23.7, 11.1, 10.2; HRMS (ESI) m/z calcd for    C₁₈H₂₃N₃O₂FS ([M+H]⁺) 364.1490, found 364.1474.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(naphthalen-1-yl)piperazin-1-yl)ethanone    (5g). A Schlenk flask was charged under N₂ with piperazine (0.0500    g, 0.580 mmol), NaO-t-Bu (0.100 g, 1.06 mmol), (rac)-BINAP (0.0051    g, 0.0079 mmol), Pd₂(dba)₃ (0.0050 g, 0.0053 mmol), and degassed    toluene (5 mL). After addition of 1-bromonaphthalene (75 μL, 0.530    mmol), the reaction mixture was heated at 110° C. for 24 h, cooled    to room temperature, diluted with CH₂Cl₂, filtered through Celite,    and concentrated in vacuo. The resulting    1-(naphthalen-1-yl)piperazine (4g) was used without further    purification for the next reaction. To a solution of    (((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0580 g,    0.272 mmol) in CH₂Cl₂ (4 mL) was added 1-(naphthalen-1-yl)piperazine    4g (0.0750 g, 0.353 mmol) and Et₃N (114 μL, 0.815 mmol). The    reaction mixture was cooled to 0° C., treated with T3P (50 wt. %    solution in EtOAc, 288 μL, 0.408 mmol), allowed to warm to room    temperature, stirred for 20 h, diluted with CH₂Cl₂, and washed with    satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and brine, dried    (Na₂SO₄), filtered, and concentrated in vacuo. The crude residue was    purified by chromatography on SiO₂ (95:5 CH₂Cl₂/MeOH) to give 5g    (0.0700 g, 0.177 mmol, 65% 2 steps, 99.9% pure by ELSD) as a yellow    oil: IR (neat) 2919, 1637, 1435, 1398, 1215, 1192 cm⁻¹; ¹H NMR (500    MHz, CDCl₃) δ 8.21 (d, 1 H, J=7.5 Hz), 7.85 (d, 1 H, J=7.5 Hz), 7.61    (d, 1 H, J=8.0 Hz), 7.54-7.49 (m, 2 H), 7.42 (d, 1 H, J=8.0 Hz),    7.08 (d, 1 H, J=7.5 Hz), 3.73-3.66 (m, 4 H), 3.64 (s, 2 H), 3.28 (s,    2 H), 3.27-2.85 (m, 4 H), 2.45 (s, 3 H), 2.32 (s, 3 H); ¹³C NMR (125    MHz, CDCl₃) δ 167.6, 166.8, 159.7, 148.7, 134.7, 128.7, 128.5,    126.0, 125.7 (2 C), 124.2, 123.0, 115.0, 109.7, 52.9, 52.7, 47.0,    42.4, 32.1, 23.7, 11.1, 10.2; HRMS (ESI) m/z calcd for C₂₂H₂₆N₃O₂S    ([M+H]⁺) 396.1746, found 396.1740.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2-methoxyphenyl)piperazin-1-yl)ethanone    (5h). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0200    g, 0.0994 mmol) in CH₂Cl₂ (1.25 mL) was added    1-(o-methoxyphenyl)piperazine (4h, 0.0230 g, 0.119 mmol) and Et₃N    (41 μL, 0.298 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 105 μL, 0.149 mmol),    warmed to room temperature, stirred for 2 d, diluted with CH₂Cl₂ and    washed with satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and brine,    dried (Na₂SO₄), filtered, and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (ISCO, 12 g column,    liquid load in CH₂Cl₂, EtOAc/hexanes gradient (10-100%, eluted at    50-70%) to give 5h (0.0195 g, 0.0519 mmol, 52%, 100% pure by ELSD)    as a colorless solid: Mp 91-93° C.; IR (ATR) 2997, 2926, 2812, 1626,    1500, 1447, 1243, 1223, 1143, 1023, 979, 751, 741, 726 cm⁻¹; ¹H NMR    (300 MHz, CDCl₃) δ 7.06-7.01 (m, 1 H), 6.95-6.87 (m, 3 H), 3.87 (s,    3 H), 3.80 (app t, 2 H, J=5.0 Hz), 3.64-3.62 (m, 4 H), 3.23 (s, 2    H), 3.07 (app t, 2 H, J=5.0 Hz), 3.03 (app t, 2 H, J=5.0 Hz), 2.41    (s, 3 H), 2.28 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 167.4, 166.7,    159.8, 152.2, 140.4, 123.6, 121.0, 118.4, 111.3, 109.7, 55.4, 50.7,    50.5, 46.7, 42.0, 32.1, 23.7, 11.0, 10.1; HRMS (ESI) m/z calcd for    C₁₉H₂₆N₃O₂₃S ([M+H]⁺) 376.1689, found 376.1673.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-N-methyl-N-(2-(methyl(o-tolyl)amino)ethyl)    acetamide (5i). To a solution of    2(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0608 g,    0.302 mmol) in CH₂Cl₂ (3.0 mL) was added    N,N′-dimethyl-N-(o-tolyl)ethane-1,2-diamine (4i, 0.0500 g, 0.275    mmol) and Et₃N (115 μL, 0.825 mmol). The reaction mixture was cooled    to 0° C., treated with T3P (50 wt. % solution in EtOAc, 292 μL,    0.412 mmol), warmed to room temperature, stirred for 20 h, diluted    with CH₂Cl₂, and washed with satd. aqueous NH₄Cl solution, satd.    aqueous NaHCO₃ solution, and brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (3:2, EtOAc/hexanes, base washed with 0.1%    Et₃N prior to use) to give 5i (0.0752 g, 0.207 mmol, 75%, 99.6% pure    by ELSD) as a light yellow oil: IR (ATR) 2932, 2795, 1640, 1598,    1493, 1451, 1421, 1393, 1196, 1108, 1047, 766, 738 cm⁻¹; ¹H NMR (400    MHz, CDCl₃, room temperature, mixture of rotamers coalescing in    DMSO-d₆ at 357 K) δ 7.20-7.12 (m, 2 H), 7.07-6.95 (m, 2 H), 3.59,    3.58 (2s, 2 H), 3.54 (t, 1 H, J=6.6 Hz), 3.39 (t, 1 H, J=6.6 Hz),    3.16-3.08 (m, 3 H), 2.97, 2.95 (2s, 4 H), 2.71, 2.67 (2s, 3 H), 2.38    (s, 3 H), 2.30 (s, 2 H), 2.27, 2.26 (3s, 4 H); ¹³C NMR (125 MHz,    CDCl₃, room temperature, mixture of rotamers coalescing in DMSO-d₆    at 357 K) δ 169.2, 168.8, 166.7 (2 C), 159.7, 151.7, 150.8, 133.8,    132.9, 131.4, 131.2, 126.7, 126.5, 124.0, 123.2, 120.2, 119.9,    109.8, 53.9, 53.2, 48.4, 46.4, 43.3, 42.3, 36.7, 33.8, 32.4, 31.6,    23.7, 23.4, 18.2, 18.0, 11.0 (2 C), 10.1; HRMS (ESI) m/z calcd for    C₁₉H₂₈N₃O₂S ([M+H]⁺) 362.1897, found 362.1890.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(4-(o-tolyl)-1,4-diazepan-1-yl)ethan-1-one    (BRE454-76; 5j). A solution of tert-butyl    4-(o-tolyl)-1,4-diazepane-1-carboxylate (29a, 0.0750 g, 0.258 mmol)    in THF (0.3 mL) was cooled to 0° C., treated with 4 M HCl in dioxane    (1.6 mL) and stirred at 0° C. for 2 h. The reaction mixture was    concentrated in vacuo and the yellow solid 4j was precipitated in    Et₂O, filtered off from the solution, washed with Et₂O, dried under    high vacuum, and used without further purification for the next    step.-   To a solution of 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic    acid (3a, 0.0460 g, 0.229 mmol) in CH₂Cl₂ (2.3 mL) was added    4-(o-tolyl)-1,4-diazepane hydrochloride (4j, 0.258 mmol) and Et₃N    (159 μL, 1.14 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 242 μL, 0.343 mmol),    warmed to room temperature, stirred for 20 h, diluted with CH₂Cl₂,    and washed with satd. aqueous NH₄Cl solution, satd. aqueous NaHCO₃    solution, and brine, dried (Na₂SO₄), filtered, and concentrated in    vacuo. The crude residue was purified by chromatography on SiO₂    (3:2, EtOAc/hexanes, base washed with 0.1% Et₃N) to give 5j (0.0854    g, 0.229 mmol, quant. 100% pure by ELSD) as a clear colorless oil:    IR (ATR) 2945, 2825, 1634, 1598, 1491, 1447, 1423, 1215, 1194, 1136,    915, 762, 726 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, room temperature,    mixture of rotamers) δ 7.20 (app d, 1 H, J=7.6 Hz), 7.17 (app t, 1    H, J=7.6 Hz), 7.05 (app d, 1 H, J=7.6 Hz), 7.01 (app dt, 1 H, J=7.2,    2.0 Hz), 3.82-3.78 (m, 2 H), 3.71-3.65 (m, 4 H), 3.24-3.20 (m, 3 H),    3.15 (t, 1 H, J=5.2 Hz), 3.12-3.07 (m, 2 H), 2.46 (app s, 3 H), 2.32    (2s, 6 H), 2.04 (sept, 2 H, J=6.0 Hz); ¹³C NMR (125 MHz, CDCl₃, room    temperature, mixture of rotamers) δ 168.9, 168.8, 166.9, 166.8,    159.8 (2 C), 153.4, 153.3, 132.9 (2 C), 131.1 (2 C), 126.7, 126.6,    123.6, 123.4, 120.8, 120.7, 109.9, 56.4, 55.8, 55.5, 54.9, 50.1,    47.6, 47.2, 44.9, 32.2, 32.0, 29.5, 28.2, 23.7, 18.5 (2 C), 11.1,    10.2 (2 C); HRMS (ESI) m/z calcd for C₂₀H₂₈N₃O₂S ([M+H]⁺) 374.1897,    found 374.1883.

-   1-(2,6-Dimethyl-4-(o-tolyl)piperazin-1-yl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)ethanone    (5k). A solution of (((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic    acid (3a, 0.0300 g, 0.142 mmol) in CH₂Cl₂ (2 mL) was treated with    3,5-dimethyl-1-(o-tolyl)piperazine (4k, 0.0350 g, 0.170 mmol) and    Et₃N (59 μL, 0.425 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 150 μL, 0.212 mmol),    warmed to room temperature, stirred for 20 h, diluted with CH₂Cl₂,    and washed with satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and    brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The    crude residue was purified by chromatography on SiO₂ (95:5    CH₂Cl₂/MeOH) to give 5k (0.0450 g, 0.116 mmol, 82%, 99.8% pure by    ELSD) as a light yellow oil: IR (neat) 2975, 1629, 1491, 1422, 1327,    1127 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.22-7.19 (m, 2 H), 7.06-7.02    (m, 2 H), 4.68 (brs, 1 H), 4.05 (brs, 1 H), 3.73-3.70 (m, 1 H),    3.66-3.61 (m, 1 H), 3.30-3.19 (m, 2 H), 2.98-2.96 (m, 2 H),    2.94-2.89 (m, 1 H), 2.81-2.78 (m, 1 H), 2.44 (s, 3 H), 2.41 (s, 3    H), 2.31 (s, 3 H), 1.55 (d, 3 H, J=6.0 Hz), 1.48 (d, 3 H, J=6.0 Hz);    ¹³C NMR (125 MHz, CDCl₃) δ 168.2, 166.7, 151.2, 133.3, 131.2, 126.8,    124.1, 119.6, 109.8, 57.0, 56.8, 49.8, 45.8, 32.0, 23.6, 21.6, 20.3,    18.2, 11.0, 10.1; HRMS (ESI) m/z calcd for C₂₁H₃₀N₃O₂S ([M+H]⁺)    388.2059, found 388.2053.

-   1-(3,5-Dimethyl-4-phenylpiperazin-1-yl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)ethan-1-one    (51). A solution of tert-butyl    3,5-dimethyl-4-phenylpiperazine-1-carboxylate (29b, 0.0330 g, 0.114    mmol) in THF (0.1 mL) at 0° C. was treated with 4 M HCl in dioxane    (0.70 mL) and stirred at 0° C. for 1.5 h and at room temperature for    1.5 h. The yellow solid was filtered off, washed with Et₂O, dried    under high vacuum and the resulting crude 4l was directly used for    the next step.-   To a solution of 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic    acid 3a (0.0229 g, 0.114 mmol) in CH₂Cl₂ (1.1 mL) was added    2,6-dimethyl-1-phenylpiperazine hydrochloride (4l, 0.0258 g, 0.114    mmol) and Et₃N (79 μL, 0.569 mmol). The reaction mixture was cooled    to 0° C., treated with T3P (50 wt. % solution in EtOAc, 121 μL,    0.171 mmol), warmed to room temperature, stirred for 20 h, diluted    with CH₂Cl₂, and washed with satd. aqueous NH₄Cl solution, satd.    aqueous NaHCO₃ solution, and brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (1:1, acetone/hexanes, base washed with 0.1%    Et₃N prior to use) to give 5l (0.0322 g, 0.0862 mmol, 76%, 100% pure    by ELSD) as a colorless oil: IR (ATR) 2967, 2931, 1639, 1597, 1493,    1449, 1377, 1319, 1272, 1238, 1151, 1091, 886, 771, 731, 703 cm⁻¹;    ¹H NMR (400 MHz, CDCl₃) δ 7.31 (t, 2 H, J=7.6 Hz), 7.18 (t, 1 H,    J=7.2 Hz), 7.10 (d, 2 H, J=7.6 Hz), 4.42 (ddd, 1 H, J=12.8, 4.0, 2.4    Hz), 3.70-3.60 (m, 3 H), 3.29-3.18 (m, 2 H), 3.10-2.93 (m, 3 H),    2.67 (dd, 1 H, J=13.2, 10.4 Hz), 2.43 (s, 3 H), 2.30 (s, 3 H), 0.77    (d, 3 H, J=6.4 Hz), 0.76 (d, 3 H, J=5.6 Hz); ¹³C NMR (100 MHz,    CDCl₃) δ 167.1, 166.8, 159.7, 148.5, 128.9, 126.4, 125.6, 109.8,    56.0, 55.6, 53.4, 48.7, 31.9, 23.7, 18.2, 18.2, 11.1, 10.2; HRMS    (ESI) m/z calcd for C₂₀H₂₈N₃O₂S ([M+H]⁺) 374.1897, found 374.1887.

-   1-(3,5-Dimethyl-4-(m-tolyl)piperazin-1-yl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)ethan-1-one    (5m). A solution of tert-butyl    3,5-dimethyl-4-(m-tolyl)piperazine-1-carboxylate (29c, 0.0400 g,    0.131 mmol) in THF (0.1 mL) at 0° C. was treated with 4 M HCl in    dioxane (0.80 mL), and stirred at 0° C. for 1.5 h and at room    temperature for 1.5 h. A yellow precipitate formed and the solid was    filtered off, washed with Et₂O, and dried under high vacuum and the    resulting crude 4m was used directly for the next step.-   To a solution of 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic    acid (3a, 0.0264 g, 0.131 mmol) in CH₂Cl₂ (1.3 mL) was added    2,6-dimethyl-1-(m-tolyl)piperazine hydrochloride (4m, 0.0316 g,    0.131 mmol) and Et₃N (91 μL, 0.656 mmol). The reaction mixture was    cooled to 0° C., treated with T3P (50 wt. % solution in EtOAc, 139    μL, 0.197 mmol), warmed to room temperature, stirred for 20 h,    diluted with CH₂Cl₂, washed with satd. aqueous NH₄Cl solution, satd.    aqueous NaHCO₃ solution, and brine, dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (3:2, EtOAc/hexanes, base washed with 0.1%    Et₃N prior to use) to give 5m (0.0400 g, 0.103 mmol, 79%, 100% pure    by ELSD) as a clear colorless oil: IR (ATR) 2966, 2929, 1637, 1602,    1451, 1376, 1319, 1271, 1194, 1149, 1108, 1088, 911, 889, 788, 730,    709 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.18 (t, 1 H, J=7.6 Hz), 6.97    (d, 1 H, J=7.6 Hz), 6.90-6.88 (m, 2 H), 4.41 (app d, 1 H, J=12.8    Hz), 3.64 (brs, 3 H), 3.27-3.19 (m, 2 H), 3.15-2.91 (m, 3 H), 2.67    (t, 1 H, J=9.2 Hz), 2.43 (s, 3 H), 2.32 (s, 3 H), 2.30, (s, 3 H),    0.77 (br app s, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 166.8,    159.7, 148.4, 138.7, 128.7, 127.1, 126.4, 123.4, 109.8, 56.0, 55.6,    53.4, 48.7, 32.0, 23.7, 21.4, 18.3, 18.2, 11.1, 10.2; HRMS (ESI) m/z    calcd for C₂₁H30N₃O₂S ([M+H]⁺) 388.2053, found 388.2046.

-   3,5-Dimethyl-4-(42-(4-(o-tolyl)piperazin-1-yl)ethyl)thio)methyl)isoxazole    (6). A solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(o-tolyl)piperazin-1-yl)ethanone    (5b, 0.0387 g, 0.108 mmol) in THF (1 mL) at 0° C. was treated with    LiAlH₄ (1 M solution in Et₂O, 120 μL, 0.118 mmol), stirred at 0° C.    for 1 h, and then quenched with Rochelle's salt (NaKC₄H₄O₆, satd.    aqueous solution, 1 mL). The mixture was stirred for an additional 1    h at 0° C., diluted with EtOAc, extracted with EtOAc (2×15 mL),    dried (Na₂SO₄), filtered, and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (ISCO, 4 g column,    liquid load in CH₂Cl₂, 0-20% MeOH/CH₂Cl₂, product eluted at 5% MeOH)    to give a colorless oil. This oil was further purified by    chromatography on SiO₂ (CH₂Cl₂ to 5:95, MeOH/CH₂Cl₂) on a pipette    column to give 6 (0.0155 g, 0.0449 mmol, 42%, 100% pure by ELSD) as    a colorless oil: IR (neat) 3393, 2925, 2814, 1637, 1599, 1493, 1448,    1424, 1372, 1227, 1195, 1130, 1041, 1006, 931, 763, 723 cm⁻¹; ¹H NMR    (300 MHz, CDCl₃) δ 7.16 (app t, 2 H, J=7.4 Hz), 7.03-6.95 (m, 2 H),    3.75 (t, 1 H, J=5.7 Hz), 3.50 (s, 2 H), 2.93 (app t, 4 H, J=4.5 Hz),    2.63 (brs, 8 H), 2.38 (s, 3 H), 2.30 (s, 3 H), 2.29 (s, 3 H); ¹³C    NMR (75 MHz, CDCl₃) δ 165.9, 159.6, 151.4, 132.6, 131.0, 126.6,    123.2, 119.0, 110.5, 77.2, 58.1, 53.6, 51.6, 29.1, 24.0, 23.5, 17.8,    11.1, 10.2; HRMS (ESI) m/z calcd for C₁₉H₂₈ON₃S ([M+H]⁺) 346.1948,    found 346.1946.

-   2-(Benzylthio)-1-(4-(o-tolyl)piperazin-1-yl)ethanone (7). A solution    of 2-(benzylthio)acetic acid 3b (0.0440 g, 0.241 mmol) in CH₂Cl₂    (3.05 mL) was treated with 1-(o-tolyl)piperazine 4b (0.0521 g, 0.290    mmol) and Et₃N (101 μL, 0.724 mmol). The reaction mixture was cooled    to 0° C., treated with T3P (50 wt. % solution in EtOAc, 256 μL,    0.362 mmol), warmed to room temperature and stirred for 2 d. The    solution was diluted with CH₂Cl₂ and washed with satd. aqueous    NH₄Cl, satd. aqueous NaHCO₃, and brine, dried (Na₂SO₄), filtered,    and concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-100%)) to give 7 (0.0635 g, 0.187 mmol,    77%, 100% pure by ELSD) as a yellow oil: IR (ATR) 2917, 1815, 1634,    1598, 1492, 1437, 1223, 1150, 1031, 975, 761, 700 cm⁻¹; ¹H NMR (300    MHz, CDCl₃) δ 7.45-7.23 (m, 7 H), 7.06-7.01 (m, 2 H), 3.89 (s, 2 H),    3.79 (app t, 2 H, J=4.9 Hz), 3.59 (app t, 2 H, J=4.9 Hz), 3.30 (s, 2    H), 2.95-2.90 (m, 4 H), 2.37 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ    167.7, 150.9, 137.7, 132.8, 131.2, 129.3, 128.5, 127.2, 126.7,    123.8, 119.3, 51.9, 51.7, 46.9, 42.4, 36.3, 32.4, 17.8; HRMS (ESI)    m/z calcd for C₂₀H₂₅N₂OS ([M+H]⁺) 341.1682, found: 341.1674.

-   4-Phenyl-1-(4-(o-tolyl)piperazin-1-yl)butan-1-one (8). To a solution    of phenyl butanoic acid (3c, 0.0500 g, 0.305 mmol) in CH₂Cl₂ (3.05    mL) was added 1-(o-tolyl)piperazine (4b, 0.0657 g, 0.365 mmol) and    Et₃N (85 μL, 0.609 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 322 μL, 0.457 mmol),    warmed to room temperature, stirred overnight, diluted with CH₂Cl₂    and washed with satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and    brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The    crude residue was purified by chromatography on SiO₂ (ISCO, 12 g    column, liquid load in CH₂Cl₂, EtOAc/hexanes gradient (10-100%),    eluted at 30%) to give 8 (0.0863 g, 0.268 mmol, 88%, 100% pure by    ELSD) as a colorless oil: IR (ATR) 3024, 2917, 2813, 1641, 1492,    1432, 1223, 1150, 1025, 761, 722 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ    7.36-7.31 (m, 2 H), 7.27-7.18 (m, 5 H), 7.07-6.99 (m, 2 H), 3.80    (app t, 2 H, J=4.8 Hz), 3.55 (app t, 2 H, J=4.8 Hz), 2.88 (app t, 4    H, J=4.8 Hz), 2.75 (t, 2 H, J=7.5 Hz), 2.41 (t, 2 H, J=7.5 Hz), 2.36    (s, 3 H), 2.06 (ddd, 2 H, J=7.9, 7.7, 7.3 Hz); ¹³C NMR (75 MHz,    CDCl₃) δ 171.2, 150.8, 141.6, 132.6, 131.0, 128.4, 128.3, 126.6,    125.8, 123.6, 119.0, 51.9, 51.6, 45.9, 41.9, 35.2, 32.3, 26.6, 17.7;    HRMS (ESI) m/z calcd for C₂₁H₂₇N20 ([M+H]⁺) 323.2118, found:    323.2110.

-   2-(Phenylthio)-1-(4-(o-tolyl)piperazin-1-yl)ethan-1-one (9). To a    solution of 2-(phenylthio)acetic acid (3d, 0.0500 g, 0.297 mmol) in    CH₂Cl₂ (3.0 mL) was added 1-(o-tolyl)piperazine (4b, 0.0642 g, 0.357    mmol) and Et₃N (83 μL, 0.594 mmol). The mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 315 μL, 0.446 mmol),    warmed to room temperature, stirred for 3 d, diluted with CH₂Cl₂ and    washed with satd. aqueous NH₄Cl, satd. aqueous NaHCO₃, and brine.    The organic layer was dried (Na₂SO₄), filtered, and concentrated in    vacuo. The crude residue was purified by chromatography on SiO₂    (ISCO, 4 g column, liquid load in CH₂Cl₂, EtOAc/hexanes gradient    (0-30%), eluted at 20-30%) to give 9 (0.0746 g, 0.229 mmol, 77%,    100% pure by ELSD) as a clear colorless oil: IR (ATR) 3057, 2947,    2911, 2856, 2815, 1639, 1598, 1492, 1482, 1382, 1275, 1223, 1203,    1149, 1115, 1032, 974, 950, 909, 762, 738, 723, 690 cm⁻¹; ¹H NMR    (400 MHz, CDCl₃) δ 7.48 (dd, 2 H, J=7.6, 1.2 Hz), 7.34 (app t, 2 H,    J=7.6 Hz), 7.26-7.17 (m, 3 H), 7.02 (app t, 1 H, J=7.6 Hz), 6.98    (app d, 1 H, J=7.6 Hz), 3.81 (s, 2 H), 3.76 (app t, 2 H, J=4.8 Hz),    3.63 (app t, 2 H, J=4.8 Hz), 2.91 (app t, 2 H, J=4.8 Hz), 2.86 (t, 2    H, J=4.8 Hz), 2.33 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1,    150.7, 134.9, 132.7, 131.2, 130.3, 129.1, 127.0, 126.7, 123.8,    119.2, 51.9, 51.6, 47.0, 42.5, 36.7, 17.8; HRMS (ESI) m/z calcd for    C₁₉H₂₃N2OS ([M+H]⁺) 327.1526, found 327.1514.

-   3-Phenyl-1-(4-(o-tolyl)piperazin-1-yl)prop-2-yn-1-one (10). To a    solution of phenyl propiolic acid (3e, 0.200 g, 1.37 mmol) in CH₂Cl₂    (12 mL) was added 1-(o-tolyl)piperazine (4b, 0.290 g, 1.64 mmol) and    Et₃N (570 μL, 4.11 mmol). The reaction mixture was cooled to 0° C.,    treated with T3P (50 wt. % solution in EtOAc, 1.45 mL, 2.05 mmol),    warmed to room temperature, stirred for 3 d, diluted with CH₂Cl₂ (30    mL), and washed with satd. aqueous NH₄Cl (5 mL), satd. aqueous    NaHCO₃ (5 mL), and brine (5 mL), dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 24 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-100%), product eluted at 40%    EtOAc/hexanes) to give 10 (0.401 g, 1.32 mmol, 96%, >99.9% pure by    ELSD) as a colorless solid: Mp 127-129° C.; IR (neat) 3037, 2907,    2857, 2206, 1616, 1491, 1424, 1279, 1226, 1207, 1035, 923, 758, 726,    686 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.59-7.56 (m, 2 H), 7.43-7.34    (m, 3 H), 7.22-7.16 (m, 2 H), 7.05-6.99 (m, 2 H), 3.99 (app t, 2 H,    J=5.0 Hz), 3.85 (app t, 2 H, J=5.0 Hz), 2.99 (app t, 2 H, J=5.0 Hz),    2.92 (app t, 2 H, J=5.0 Hz), 2.35 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃)    δ 153.2, 150.8, 132.8, 132.4, 131.2, 130.1, 128.6, 126.8, 123.9,    120.5, 119.3, 90.9, 81.2, 52.2, 51.5, 47.7, 42.1, 17.8; HRMS (ESI)    m/z calcd for C₂₀H₂₁ON₂ ([M+H]⁺) 305.1648, found 305.1643.

-   (E)-3-Phenyl-1-(4-(o-tolyl)piperazin-1-yl)prop-2-en-1-one (11). A    solution of trans-cinnamic acid (3f, 0.0400 g, 0.270 mmol) in CH₂Cl₂    (2.5 mL) was treated with 1-(o-tolyl)piperazine (4b, 0.0570 g, 0.320    mmol), Et₃N (113 μL, 0.810 mmol). The reaction mixture was cooled to    0° C., treated with T3P (50 wt. % solution in EtOAc, 290 μL, 0.405    mmol), warmed to room temperature, stirred for 3 d, diluted with    CH₂Cl₂ (10 mL), and washed with satd. aqueous NH₄Cl (2 mL), satd.    aqueous NaHCO₃ (2 mL), and brine (2 mL), dried (Na₂SO₄), filtered,    and concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-100%), product eluted at 35%,    EtOAc/hexanes) to give 11 (0.0520 g, 0.168 mmol, 62%, >99% purity by    ELSD) as a yellow solid: Mp 110-111° C.; IR (neat) 3045, 2920, 2840,    1643, 1595, 1423, 1327, 1225, 1152, 986, 765, 710, 682 cm⁻¹; ¹H NMR    (400 MHz, CDCl₃) δ 7.72 (d, 1 H, J=11.4 Hz), 7.55 (dd, 2 H, J=6.8,    1.4 Hz), 7.41-7.36 (m, 3 H), 7.20 (dd, 2 H, J=14.6, 7.4 Hz), 7.02    (ddd, 2 H, J=14.6, 7.4, 0.6 Hz), 6.95 (d, 1 H, J=15.6 Hz), 3.90    (brs, 2 H), 3.81 (brs, 2 H), 2.96 (brs, 4 H), 2.36 (s, 3 H); ¹³C NMR    (75 MHz, CDCl₃) δ 165.5, 150.8, 142.8, 135.2, 132.7, 131.1, 129.6,    128.8, 127.7, 126.6, 123.7, 119.2, 117.1, 52.1, 51.6, 46.4, 42.6,    17.8; HRMS (ESI) m/z calcd for C₂₀H₂₃ON₂ ([M+H]⁺) 307.1805, found    307.1796.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)sulfinyl)-1-(4-(o-tolyl)piperazin-1-yl)ethan-1-one    (12). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(o-tolyl)piperazin-1-yl)ethanone    (5b, 0.0500 g, 0.139 mmol) in MeOH (0.30 mL) at 0° C. was added    dropwise a solution of sodium metaperiodate (0.0301 g, 0.139 mmol)    in water (0.14 mL). The resulting heterogeneous mixture was allowed    to warm to room temperature and stirred for 15 h. The reaction    mixture was filtered through a plug of Celite (MeOH), concentrated,    dissolved in CH₂Cl₂, dried (MgSO₄), filtered and concentrated in    vacuo. The crude residue was purified by chromatography on SiO₂    (100% EtOAc) to give 12 (0.0356 g, 0.0948 mmol, 68%, 100% pure by    ELSD) as a colorless foam: IR (ATR) 2917, 2818, 1631, 1599, 1493,    1441, 1384, 1275, 1224, 1195, 1151, 1053, 1028, 911, 764, 727 cm⁻¹;    ¹H NMR (400 MHz, CDCl₃) δ 7.21-7.15 (m, 2 H), 7.02 (app t, 1 H,    J=7.2 Hz), 6.97 (app d, 1 H, J=8.0 Hz), 4.18 (d, 1 H, J=14.0 Hz),    3.90-3.84 (m, 5 H), 3.64 (app t, 2 H, J=4.4 Hz), 2.95 (app t, 2 H,    J=4.4 Hz), 2.85 (brs, 2 H), 2.45 (s, 3 H), 2.32 (s, 3 H), 2.31 (s, 3    H); ¹³C NMR (100 MHz, CDCl₃) δ 169.2, 162.9, 159.9, 150.4, 132.7,    131.2, 126.7, 124.0, 119.2, 104.5, 53.7, 52.0, 51.5, 47.0, 46.8,    42.5, 17.7, 11.6, 10.3; HRMS (ESI) m/z calcd for C₁₉H₂₆N303S    ([M+H]⁺) 376.1689, found 376.1684.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)sulfonyl)-1-(4-(o-tolyl)piperazin-1-yl)ethan-1-one    (13). A solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(o-tolyl)piperazin-1-yl)ethanone    (5b, 0.0429 g, 0.117 mmol) in CH₂Cl₂ (0.65 mL) was treated with    3-chloroperoxybenzoic acid (70 wt. %, 0.0576 g, 0.234 mmol) in 2    portions. The reaction mixture was stirred at room temperature for    15 h, quenched with 10% aqueous sodium metabisulfite solution (2    mL), diluted with aqueous 1 M NaOH (10 mL) and extracted with CH₂Cl₂    (2×15 mL). The combined organic layers were washed with 1 M NaOH (10    mL), dried (MgSO₄), filtered, and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (70-100%    EtOAc/hexanes) to give 13 (0.0203 g, 0.0519 mmol, 44%, 100% pure by    ELSD) as a colorless foam: IR (ATR) 2919, 2819, 1641, 1599, 1493,    1445, 1318, 1225, 1150, 1126, 1030, 911, 765, 728 cm⁻¹; ¹H NMR (400    MHz, CDCl₃) δ 7.21-7.16 (m, 2 H), 7.05-6.98 (m, 2 H), 4.36 (s, 2 H),    4.09 (s, 2 H), 3.85 (app brs, 2 H), 3.72 (brt, 2 H, J=4.0 Hz), 3.00    (brt, 2 H, J=4.0 Hz), 2.93 (brt, 2 H, J=4.4 Hz), 2.50 (s, 3 H), 2.35    (s, 3 H), 2.33 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 160.7,    160.3, 150.3, 132.7, 131.2, 126.7, 124.0, 119.2, 101.8, 54.9, 51.7,    51.4, 48.3, 47.8, 43.0, 17.8, 11.5, 10.2; HRMS (ESI) m/z calcd for    C₁₉H₂₆N₃O₄S ([M+H]⁺) 392.1639, found 392.1633.

-   (Z)-3-Phenyl-1-(4-(o-tolyl)piperazin-1-yl)prop-2-en-1-one (14). To a    solution of 3-phenyl-1-(4-(o-tolyl)piperazin-1-yl)prop-2-yn-1-one    (10, 0.103 g, 0.337 mmol) in MeOH (2 mL) and EtOAc (1 mL) was added    Lindlar's catalyst (5% Pd on CaCO₃, lead poisoned, 0.120 g) and    quinoline (15 μL, 0.130 mmol). The reaction mixture was purged and    backfilled with H₂ (balloon, 2×), allowed to stir for 45 min,    filtered through SiO₂, and concentrated in vacuo. The crude residue    was purified by chromatography on SiO₂ (ISCO, modified dry load in    CH₂Cl₂, 0-90% EtOAc/hexanes gradient, product eluted at 25%    EtOAc/hexanes) to give 14 (0.104 g, 0.339 mmol, quant., 99.6% purity    by ELSD) as a yellow oil: IR (neat) 3022, 2914, 2815, 1513, 1597,    1493, 1434, 1364, 1223, 1149, 1115, 1034, 973, 913, 855, 762, 722,    698 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.41-7.30 (m, 5 H), 7.17-7.11    (m, 2 H), 6.98 (t, 1 H, J=7.1 Hz), 6.81 (d, 1 H, J=7.8 Hz), 6.71 (d,    1 H, J=12.6 Hz), 6.07 (d, 1 H, J=12.6 Hz), 3.81 (app brt, 2 H, J=4.8    Hz), 3.48 (app t, 2 H, J=4.8 Hz), 2.81 (app t, 2 H, J=4.8 Hz), 2.44    (app t, 2 H, J=4.8 Hz), 2.25 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ    167.6, 150.9, 135.6, 133.5, 132.7, 131.1, 128.7, 128.6, 128.5,    126.6, 123.7, 123.2, 119.1, 51.5, 51.3, 46.8, 41.7, 17.7; HRMS (ESI)    m/z calcd for C₂₀H₂₃ON₂ ([M+H]⁺) 307.1805, found 307.1800.

-   (2-Phenylcyclopropyl)(4-(o-tolyl)piperazin-1-yl)methanone (15). A    solution of anhydrous CrCl₂ (0.0486 g, 0.392 mmol) in THF (0.6 mL)    at room temperature under N₂ was treated with a solution of    (Z)-3-phenyl-1-(4-(o-tolyl)piperazin-1-yl)prop-2-en-1-one (14,    0.0200 g, 0.0653 mmol) in THF (0.5 mL) and CH₂ICl (20 μL, 0.261    mmol). The reaction mixture was stirred for 18 h at reflux, quenched    by addition of 1 M aqueous HCl (6 mL) and extracted with EtOAc. The    combined organic layers were dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (4:1, EtOAc/hexanes) to give 15 (0.0120 g,    0.0375 mmol, 57%, 100% pure by ELSD) as a brown oil: IR (neat) 2920,    1638, 1491, 1457, 1340, 1223, 1028 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ    7.30-7.27 (m, 2 H), 7.22-7.11 (m, 5 H), 6.98 (dt, 1 H, J=7.2, 1.2    Hz), 6.72 (dd, 1 H, J=7.9, 0.8 Hz), 3.93-3.90 (m, 1 H), 3.77-3.73    (m, 1 H), 3.60-3.53 (m, 1 H), 3.30-3.22 (m, 1 H), 2.75-2.72 (m, 2    H), 2.50-2.41 (m, 1 H), 2.26 (s, 3 H), 2.24-2.16 (m, 1 H), 2.10-2.00    (m, 1 H), 1.87 (dd, 1 H, J=12.4, 5.8 Hz), 1.40-1.33 (m, 1 H),    0.92-0.80 (m, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 150.9, 137.6,    132.7, 131.0, 128.2, 127.4, 126.5, 126.4, 123.6, 119.2, 51.9, 51.6,    45.7, 42.3, 24.4, 24.1, 17.7, 10.60 HRMS (ESI) m/z calcd for    C₂₁H₂₅ON₂ ([M+H]⁺) 321.1967, found 321.1961.

-   ((1SR,2SR)-2-Phenylcyclopropyl)(4-(o-tolyl)piperazin-1-yl)methanone    (16). To a solution of trans-2-phenylcyclopropanecarboxylic acid    (3g, 0.0400 g, 0.247 mmol) in CH₂Cl₂ (2.5 mL) was treated with    1-(o-tolyl)piperazine (4b, 0.0540 g, 0.296 mmol), Et₃N (100 μL,    0.740 mmol). The reaction mixture was cooled to 0° C., treated with    T3P (50 wt. % solution in EtOAc, 260 μL, 0.370 mmol, 1.5 equiv),    warmed to room temperature, stirred for 3 d, diluted with EtOAc (10    mL), and washed with satd. aqueous NH₄Cl (2 mL), satd. aqueous    NaHCO₃ (2 mL), and brine (2 mL), dried (Na₂SO₄), filtered, and    concentrated in vacuo. The crude residue was purified by    chromatography on SiO₂ (ISCO, 12 g column, liquid load in CH₂Cl₂,    EtOAc/hexanes gradient (10-90%), product eluted at 20%) to give 16    (0.0676 g, 0.211 mmol, 86%, >99.9% pure by ELSD) as a yellow oil: IR    (neat) 3026, 2912, 2814, 1631, 1600, 1493, 1440, 1381, 1223, 1150,    1033, 919, 910, 760, 723, 696 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ    7.32-7.26 (m, 2 H), 7.23-7.12 (m, 5 H), 7.01 (dd, 2 H, J=11.1, 7.5    Hz), 3.79 (brs, 4 H), 2.90 (brs, 4 H), 2.52 (brpent, 1 H, J=4.6 Hz),    2.33 (s, 3 H), 2.02 (pent, 1 H, J=4.6 Hz), 1.71 (pent, 1 H, J=4.6    Hz), 1.34-1.26 (m, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 170.6, 150.9,    141.0, 132.7, 131.2, 128.6, 126.7, 126.3, 126.1, 123.8, 119.2, 52.2,    51.7, 46.2, 25.6, 23.3, 17.9, 16.2; HRMS (ESI) m/z calcd for    C₂₁H₂₅ON₂ ([M+H]⁺) 321.1961, found 321.1957.

-   2-Chloro-1-(4-(o-tolyl)piperazin-1-yl)ethanone (17a) (Glennon et    al., J. Med. Chem. 1986, 29, 2375-2380; Jorand-Lebrun et al., J.    Med. Chem. 1997, 40, 3974-3978.). To a solution of chloroacetyl    chloride (0.698 g, 6.05 mmol) and potassium carbonate (1.14 g, 8.25    mmol) in THF (7.0 mL) was added 1-(o-tolyl)piperazine (4b, 1.00 g,    5.50 mmol) in THF (12.6 mL) at 0° C. The reaction mixture was    gradually warmed to room temperature, stirred for 16 h, diluted with    water, and extracted with EtOAc (3×20 mL). The combined organic    extracts were washed sequentially with satd. aqueous NaHCO₃, 0.1 M    aqueous HCl, and brine, dried (Na₂SO₄), filtered and concentrated in    vacuo. The crude solid was filtered through a plug of SiO₂ (3:7,    EtOAc/hexanes v/v 1% Et₃N) and washed thoroughly with EtOAc/hexanes    (3:7) to give 17a (1.37 g, 5.42 mmol, 99%) as an off white solid: ¹H    NMR (400 MHz, CDCl₃) δ 7.22-7.16 (m, 2 H), 7.05-6.99 (m, 2 H), 4.12    (s, 2 H), 3.78 (app t, 2 H, J=4.8 Hz), 3.67 (app t, 2 H, J=4.8 Hz),    2.97 (app t, 2 H, J=4.8 Hz), 2.91 (app t, 2 H, J=4.8 Hz), 2.33 (s, 3    H).

-   1-((Chloromethyl)sulfonyl)-4-(o-tolyl)piperazine (17b) (Zhou et    al., J. Org. Lett. 2008, 10, 2517-2520.). To a solution of    1-(o-tolyl)piperazine (4b, 0.500 g, 2.75 mmol) in CH₂Cl₂ (9.8 mL)    and Et₃N (0.390 mL, 2.75 mmol) at 0° C. was added    chloromethanesulfonyl chloride (0.460 g, 3.03 mmol). The reaction    mixture was stirred at 0° C., gradually warmed to room temperature    quenched after 14 h with satd. aqueous NH₄Cl solution (3 mL), and    extracted with EtOAc (3×20 mL). The combined organic extracts were    washed water (2×10 mL) and brine (10 mL), dried (Na₂SO₄), filtered    and concentrated in vacuo. The crude solid was filtered through a    plug of SiO₂ (3:7, EtOAc/hexanes containing 1% Et₃N) and washed    thoroughly with EtOAc/hexanes (3:7). The combined filtrates were    concentrated in vacuo to give 17b (0.676 g, 2.34 mmol, 85%) as an    orange solid: ¹H NMR (300 MHz, CDCl₃) δ 7.19 (t, 2 H, J=8.1 Hz),    7.03 (t, 2 H, J=8.1 Hz), 4.56 (s, 2 H), 3.63 (app t, 4 H, J=5.0 Hz),    2.99 (app t, 4 H, J=5.0 Hz), 2.32 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃)    δ 150.6, 132.7, 131.2, 126.8, 124.1, 119.4, 54.5, 51.9, 47.1, 17.7.

-   2-((3,5-Dimethylisoxazol-4-yl)methoxy)-1-(4-(o-tolyl)piperazin-1-yl)ethan-1-one    (18a). A solution of (3,5-dimethylisoxazol-4-yl)methanol (27, 0.0302    g, 0.237 mmol) in THF (0.48 mL) was cooled to 0° C. and NaH (60%    dispersion in mineral oil, 0.0190 g, 0.475 mmol) was added. The    reaction mixture was stirred at 0° C. for 30 min, treated with    2-chloro-1-(4-(o-tolyl)piperazin-1-yl)ethanone (17a, 0.0600 g, 0.237    mmol), warmed to room temperature, stirred for 20 h, quenched with    brine (1 mL), diluted with EtOAc (15 mL) and brine (5 mL), and    extracted with EtOAc (2×15 mL). The combined organic layers were    dried (Na₂SO₄) and concentrated in vacuo. The crude residue was    purified by chromatography on SiO₂ (3:2, EtOAc/hexanes) to give 18a    (0.0735 g, 0.214 mmol, 90%, 100% pure by ELSD) as a light yellow    oil: IR (ATR) 2918, 2817, 1645, 1599, 1493, 1443, 1369, 1273, 1225,    1116, 1030, 977, 764, 725 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.21-7.16    (m, 2 H), 7.02 (dt, 1 H, J=7.6, 1.2 Hz), 6.97 (app d, 1 H, J=8.0    Hz), 4.41 (s, 2 H), 4.17 (s, 2 H), 3.77 (brs, 2 H), 3.59 (app t, 2    H, J=4.8 Hz), 2.89 (app t, 4 H, J=3.6 Hz), 2.41 (s, 3 H), 2.32 (s, 3    H), 2.30 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 167.8, 167.5, 159.8,    150.7, 132.7, 131.2, 126.7, 123.9, 119.2, 110.5, 68.7, 61.7, 52.1,    51.7, 45.6, 42.3, 17.8, 11.1, 10.1; HRMS (ESI) m/z calcd for    C₁₉H₂₆N₃O₃ ([M+H]⁺) 344.1969, found 344.1960.

-   2-(Benzyl(methyl)amino)-1-(4-(o-tolyl)piperazin-1-yl)ethanone (18b).    A solution of 2-chloro-1-(4-(o-tolyl)piperazin-1-yl)ethanone (17a,    0.0534 g, 0.211 mmol), in CH₃CN (4 mL) was treated with    N-methylbenzylamine (23 μL, 0.176 mmol) and K₂CO₃ (0.730 g, 0.528    mmol). The reaction mixture was heated at reflux for 5 h, cooled to    room temperature, filtered, and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (2:3, EtOAc/hexanes)    to give 18b (0.0590 g, 0.175 mmol, 99%, >95% pure by LCMS) as a    light yellow oil: IR (neat) 2933, 2816, 1640, 1450, 1491, 1222 cm⁻¹;    ¹H NMR (500 MHz, CDCl₃) δ 7.39-7.33 (m, 4 H), 7.31-7.27 (m, 1 H),    7.23-7.19 (m, 2 H), 7.04 (t, 1 H, J=7.5 Hz), 7.01 (d, 1 H, J=8.0    Hz), 3.77 (brs, 2 H), 3.71-3.69 (m, 2 H), 3.61 (s, 2 H), 3.27 (s, 2    H), 2.91-2.87 (m, 4 H), 2.35 (s, 3 H) 2.34 (s, 3 H); ¹³C NMR (125    MHz, CDCl₃) δ 150.9, 138.1, 132.6, 131.1, 129.1, 128.2, 127.2,    126.6, 123.6, 119.1, 62.0, 60.3, 52.1, 51.7, 46.1, 42.4, 42.2, 17.8;    HRMS (ESI) m/z calcd for C₂₁H₂₈N30 ([M+H]⁺) 338.2238, found    338.2211.

-   3,5-Dimethyl-4-(((((4-(o-tolyl)piperazin-1-yl)sulfonyl)methyl)thio)methyl)isoxazole    (18c). A suspension of NaH (60% dispersion in mineral oil, 0.0200 g,    0.499 mmol) in THF (0.6 mL) was treated under an atmosphere of N₂ at    0° C. with a solution of (3,5-dimethylisoxazol-4-yl)methanethiol    (25, 0.0536 g, 0.374 mmol) in THF (0.4 mL). The reaction mixture was    stirred for 10 min, treated with    1-((chloromethyl)sulfonyl)-4-(o-tolyl)piperazine (17b, 0.0360 g,    0.125 mmol), stirred for 2 d at room temperature, quenched (water)    and extracted (EtOAc). The combined organic layers were dried    (Na₂SO₄), filtered, and concentrated in vacuo. The residue was    purified by chromatography on SiO₂ (1:4, EtOAc/hexanes) to give    crude 18c that was further purified by preparative TLC (2:3,    Et₂O/hexanes) to give 18c (2.0 mg, 0.00506 mmol, 4%, 100% pure by    ELSD) as a colorless oil: IR (neat) 2924, 1636, 1450, 1420, 1320,    1152 cm ¹; ¹H NMR (400 MHz, CDCl₃) δ 7.20 (t, 2 H, J=7.7 Hz),    7.06-7.00 (m, 2 H), 3.87 (s, 2 H), 3.76 (s, 2 H), 3.58 (app t, 4 H,    J=4.8 Hz), 2.99 (app t, 4 H, J=4.8 Hz), 2.44 (s, 3 H), 2.32 (s, 3    H), 2.31 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 159.7, 150.6,    132.7, 131.2, 126.8, 124.0, 119.4, 108.7, 51.8, 48.6, 47.0, 24.1,    17.8, 11.1, 10.2; HRMS (ESI) m/z calcd for C₁₈H₂₆O₃N₃S₂ ([M+H]⁺)    396.1416, found 396.1410.

-   N-((((3,5-Dimethylisoxazol-4-yl)methyl)thio)methyl)-4-(o-tolyl)piperazine-1-carboxamide    (20a). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0500    g, 0.248 mmol) in toluene (4.0 mL) was added DPPA (57 μL, 0.261    mmol) and Et₃N (37 μL, 0.261 mmol). The reaction mixture was heated    at 110° C. for 60 min, cooled and washed with satd. aqueous NaHCO₃,    dried (MgSO₄), filtered and concentrated to give the isocyanate 19    as a pink oil that was used without further purification.-   A solution of 1-(o-tolyl)piperazine (4b, 0.460 g, 0.261 mmol) and    Et₃N (37 μL, 0.261 mmol) in CH₂Cl₂ (0.5 mL) was cooled to 0° C. and    treated with a solution of the isocyanate 19 in CH₂Cl₂ (0.5 mL). The    reaction mixture was stirred overnight at room temperature, then    diluted with EtOAc and satd. aqueous NH₄Cl. The organic layer was    washed with satd. aqueous NaHCO₃ and brine, dried (Na₂SO₄),    filtered, and concentrated in vacuo. The residue was purified by    chromatography on SiO₂ (ISCO, 4 g column, gradient hexanes to 1:1,    EtOAc/hexanes, with an initial base wash of the column using hexanes    containing 1% Et₃N) to give 20a (0.0606 g, 0.162 mmol, 65%, 98% pure    by ELSD) as a clear oil that turns to a red oil upon standing: IR    (CH₂Cl₂) 3336, 2941, 2891, 2850, 1629, 1523, 1491, 1495, 1420, 1254,    1223, 1193, 997, 907, 761, 731 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.18    (dd, 2 H, J=8.7, 7.5 Hz), 7.04-6.98 (m, 2 H), 4.88 (brt, 1 H, J=6.0    Hz), 4.44 (d, 2 H, J=6.0 Hz), 3.67 (s, 2 H), 3.50 (app t, 4 H, J=5.0    Hz), 2.89 (app t, 4 H, J=5.0 Hz), 2.39 (s, 3 H), 2.32 (s, 3 H), 2.29    (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 166.0, 159.5, 156.9, 150.9,    132.7, 131.2, 126.7, 123.7, 119.1, 110.8, 51.6, 44.4, 43.9, 23.6,    17.8, 11.0, 10.2; HRMS (ESI) m/z calcd for C₁₉H₂₇N₄O₂S ([M+H]⁺)    375.1849, found 375.1845.

-   1-(o-Tolyppiperidin-4-yl((((3,5-dimethylisoxazol-4-yl)methyl)thio)methyl)-carbamate    (20b). To a solution of    2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)acetic acid (3a, 0.0500    g, 0.248 mmol) in toluene (4.0 mL) was added DPPA (0.06 mL, 0.261    mmol) and Et₃N (37 μL, 0.261 mmol). The reaction mixture was heated    at 110° C. for 60 min, cooled to room temperature and treated with a    solution of 1-(o-tolyl)piperidin-4-ol (4n, 0.0427 g, 0.224 mmol) in    CH₂Cl₂ (0.5 mL). The reaction mixture was stirred overnight at 80°    C., and diluted with EtOAc and satd. aqueous NH₄Cl. The organic    layer was washed with satd. aqueous NaHCO₃ and brine, dried    (Na₂SO₄), filtered, and concentrated in vacuo. The residue was    purified by chromatography on SiO₂ (ISCO, 4 g column, gradient    hexanes to 3:7, EtOAc/hexanes, with an initial base wash of the    column with hexanes w/1% Et₃N) to give 20b (0.0168 g, 0.0431 mmol,    17%, 100% pure by ELSD) as a clear oil that eventually turned to a    light yellow oil upon standing: IR (CH₂Cl₂) 3323, 2947, 2924, 2848,    2811, 1711, 1491, 1450, 1422, 1228, 1195, 1027, 762, 723 cm⁻¹; ¹H    NMR (400 MHz, DMSO-d6) δ 7.98 (t, 1 H, J=6.4 Hz), 7.16-7.11 (m, 2    H), 7.02 (d, 1 H, J=7.2 Hz), 6.94 (dt, 1 H, J=7.2, 1.2 Hz),    4.72-4.69 (m, 1 H), 4.15 (d, 2 H, J=6.4 Hz), 3.66 (s, 2 H),    3.01-2.98 (m, 2 H), 2.78-2.72 (m, 2 H), 2.36 (s, 3 H), 2.24 (s, 3    H), 2.18 (s, 3 H), 2.04-1.94 (m, 2 H), 1.77-1.67 (m, 2 H); ¹³C NMR    (100 MHz, DMSO-d6) δ 165.7, 159.2, 155.5, 151.5, 131.8, 130.7,    126.5, 122.8, 118.9, 110.9, 69.9, 49.2, 42.9, 31.6, 21.9, 17.4,    10.5, 9.7; HRMS (ESI) m/z calcd for C₂₀H₂₈N₃O₃S ([M+H]⁺) 390.1846,    found 390.1846.

-   tert-Butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (21a) (WO    2012/152854 A1). A solution of nortropinoneHCl (21, 2.00 g, 12.4    mmol) in a minimum amount of water (6.0 mL) was cooled to 0° C.,    treated dropwise with 1 M NaOH (14.8 mL, 14.8 mmol, 1.2 equiv),    warmed to room temperature over 20 min, extracted with CH₂Cl₂ (3×40    mL), dried (MgSO₄), filtered, and concentrated in vacuo (water bath    at 23° C.) to give nortropinone 21 as the free base (1.54 g,    quant.). The colorless oil was used without further purification.-   To a solution of nortropinone 21 (1.54 g, 12.3 mmol) in CH₂Cl₂ (50    mL) cooled to 0° C. was added Boc anhydride (4.26 mL, 18.6 mmol),    DMAP (0.302 g, 2.47 mmol), and Et₃N (7.0 mL, 50.2 mmol). The    reaction mixture was allowed to warm to room temperature and stirred    overnight. After 19 h, the reaction mixture was concentrated in    vacuo, and the residue was diluted with water, extracted with EtOAc    (3×), washed with brine, dried (MgSO₄), filtered, and concentrated    in vacuo to give a red sticky solid which was purified by    chromatography on SiO₂ (CH₂Cl₂) to give 21a (2.18 g, 9.68 mmol, 78%    over two steps) as a pale yellow oil that solidified to an off-white    solid upon standing at room temperature: ¹H NMR (300 MHz, DMSO-d6) δ    4.34-4.30 (m, 2 H), 2.55 (dt, 2 H, J=15.6, 4.2 Hz), 2.23 (d, 2 H,    J=15.6 Hz), 2.20 (app s, 1 H), 2.03-1.94 (m, 2 H), 1.60-1.52 (m, 2    H), 1.44 (s, 9 H); ¹³C NMR (75 MHz, DMSO-d6) δ 207.4, 152.6, 79.2,    52.7, 48.1, 28.0 (2 C).

-   tert-Butyl    3-(((trifluoromethyl)sulfonyl)oxy)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (22)    (WO 2012/152854 A1). A solution of NaHMDS (0.895 g, 4.88 mmol) in    THF (12 mL) was added dropwise (over 10 min) at −78° C. to a    solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate    (21a, 1.00 g, 4.44 mmol) in THF (12 mL). The reaction mixture was    stirred at −78° C. for 2 h, treated dropwise (over 20 min) with a    solution of PhN(Tf)₂ (1.90 g, 5.33 mmol) in THF (12 mL), stirred for    an additional 30 min at −78° C. and then allowed to warm to room    temperature and stirred for 2 h. After addition of 10% aqueous    Na₂CO₃ (50 mL), the solution was extracted with Et₂O (2×75 mL). The    combined organic layers were washed with 10% aqueous Na₂CO₃    solution, dried (MgSO₄), and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (1:19, EtOAc/hexanes    with 1% Et₃N) to give 22 (1.24 g, 3.47 mmol, 78%) as a clear oil    that solidified to a wax upon storage at −20° C.: ¹H NMR (400 MHz,    CDCl₃) δ 6.09 (brs, 1 H), 4.54-4.38 (m, 2 H), 3.07-3.02 (m, 1 H),    2.30-2.20 (m, 1 H), 2.11-1.99 (m, 3 H), 2.00-1.97 (m, 2 H),    1.79-1.70 (m, 1 H), 1.45 (s, 9 H).

-   tert-Butyl 3-(o-tolyl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate    (23a). A solution of Na₂CO₃ (0.330 g, 3.11 mmol), lithium chloride    (0.0600 g, 1.41 mmol), tert-butyl    3-(((trifluoromethyl)sulfonyl)oxy)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate    (22, 0.460 g, 1.41 mmol) and o-tolylboronic acid (0.235 g, 1.70    mmol) in DME (11 mL) and H₂O (3 mL) was sparged with N₂ for 1 h, and    treated with Pd(PPh₃)₄ (0.0376 g, 0.0325 mmol). The flask was    evacuated and backfilled with nitrogen (3×) and the mixture was    heated at 60° C. for 3 h. The mixture was allowed to cool to room    temperature, diluted with brine, extracted with EtOAc (3×), dried    (Na₂SO₄), and concentrated in vacuo. The resulting brown oil was dry    loaded onto SiO₂ and purified by chromatography on SiO₂ (hexanes to    15:1, hexanes/EtOAc) to give 23a (0.330 g, 1.10 mmol, 78%) as a    colorless solid: Mp 67.5-68.4° C.; IR (neat) 2975, 2934, 1685, 1420,    1364, 1329, 1169, 1094 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, mixture of    rotamers) δ 7.20-7.12 (m, 3 H), 7.02-7.00 (m, 1 H), 5.94-5.86 (m, 1    H), 4.50-4.30 (m, 2 H), 3.11-2.91 (m, 1 H), 2.27 (app s, 4 H),    2.10-1.90 (m, 3 H), 1.90-1.80 (m, 1 H), 1.50 (s, 9 H); ¹³C NMR (100    MHz, CDCl₃, 1:1 mixture of rotamers) δ 154.4, 141.6, 136.2, 135.5,    134.9, 131.3, 130.8, 130.7, 130.1, 129.3, 128.1, 126.9, 126.8,    125.6, 123.5, 120.0, 114.8, 79.3, 53.6, 52.9, 52.7, 52.0, 39.2,    38.4, 34.9, 34.3, 30.4, 29.6, 28.4, 19.5, 15.8; HRMS (ESI) m/z calcd    for C₁₄H₁₇N ([M+H—C₅H₉O₂]⁺) 200.1439, found 200.1435.

-   2-Chloro-1-(3-(o-tolyl)-8-azabicyclo[3.2.1]octan-8-yl)ethan-1-one    (24). A solution of tert-butyl    3-(o-tolyl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (23a, 0.196    g, 0.655 mmol) in EtOH (5.0 mL) was treated with Pd/C (5%, 0.0480    g). The flask was evacuated and flushed with H₂ (balloon, 3×). The    reaction mixture was stirred under H₂ (1 atm, balloon) overnight,    filtered through Celite, rinsed with EtOH and concentrated in vacuo    to give (23, 0.160 g, 0.531 mmol, 81%) as a yellow liquid that was    used without further purification.

A solution of 23 (0.200 g, 0.664 mmol) in CH₂Cl₂ (5 mL) was treated atroom temperature with TFA (0.30 mL, 3.98 mmol). After 16 h, the solutionwas concentrated in vacuo. The oily residue was extracted with CH₂Cl₂,washed with satd. aqueous NaHCO₃ and brine, dried (Na₂SO₄), filtered,and concentrated in vacuo to give 3-(o-tolyl)-8-azabicyclo[3.2.1]octane(23b, 0.133 g, 0.661 quant) as a light yellow oil that was used withoutfurther purification.

A solution of 23b (0.130 g, 0.646 mmol) and Et₃N (0.10 mL, 0.710 mmol)in THF (3 mL) was cooled to 0° C. and treated with chloroacetyl chloride(60 μL, 0.710 mmol) dropwise over 1 min. The reaction mixture wasstirred at 0° C. for 1 h and then at room temperature for 20 h. Thesolution was filtered, concentrated in vacuo and the residue wasdissolved in EtOAc, washed with water, dried (Na₂SO₄), filtered, andconcentrated in vacuo. The crude residue was purified by chromatographyon SiO₂ (1:1, hexanes/EtOAc) to give 24 (0.141 g, 0.508 mmol, 79%) as abrown oil. ¹H NMR analysis indicated an approximately 4:3 ratio of endolexo isomers: ¹H NMR (400 MHz, CDCl₃) δ 7.22-7.09 (m, 6.8 H), 4.85-4.80(m, 1 H), 4.80-4.74 (m, 0.7 H), 4.38-4.30 (m, 1.7 H), 4.14-4.04 (m, 3.6H), 3.49-3.39 (m, 1 H), 2.99-2.88 (m, 0.7 H), 2.58-2.49 (m, 1 H), 2.38(s, 3 H), 2.32 (s, 2 H), 2.22-1.70 (m, 11 H), 1.55-1.48 (m, 1 H); ¹³CNMR (100 MHz, CDCl₃) δ 163.4, 162.1, 141.8, 141.7, 135.9, 135.0, 130.4(2 C), 126.5, 126.4, 126.2, 126.1 (2 C), 126.0, 55.7, 55.4, 52.6, 49.6,41.5, 41.4, 39.5, 39.1, 37.9, 37.5, 32.8, 30.9, 30.3, 29.7, 28.9, 27.1,19.4, 19.3; HRMS (ESI) m/z calcd for C₁₆H₂₁ClNO ([M+H]⁺), 298.1312,found 298.1301.

-   2-(((3,5-Dimethylisoxazol-4-yl)methyl)thio)-1-(3-endo-(o-tolyl)-8-azabicyclo[3.2.1]octan-8-yl)ethanone    (26a) and    2-4(3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(3-exo-(o-tolyl)-8-azabicyclo[3.2.1]octan-8-yl)ethanone    (26b). A solution of (3,5-dimethylisoxazol-4-yl)methanethiol (25,    0.0247 g, 0.172 mmol) in THF (0.4 mL) was added to a suspension of    NaH (60% dispersion in mineral oil, 0.0115 g, 0. mmol) in THF (1.0    mL) at 0° C. The resultant slurry was stirred at 0° C. for 30 min    and a solution of    2-chloro-1-(3-(o-tolyl)-8-azabicyclo[3.2.1]octan-8-yl)ethanone (24,    0.0400 g, 0.144 mmol) in THF (0.4 mL) was added. The reaction    mixture was allowed to warm to room temperature, stirred for 24 h,    quenched with brine (1 mL), diluted with EtOAc (15 mL) and brine (5    mL), and extracted with EtOAc (2×15 mL). The combined organic layers    were dried (Na₂SO₄) and concentrated in vacuo. The crude residue was    purified by chromatography on SiO₂ (3:7, EtOAc/hexanes) to give 26a    (16.2 mg, 0.0421 mmol, 29%, 99.8% pure by ELSD) and 26b (16.6 mg,    0.0432 mmol, 30%, 100% pure by ELSD) as light yellow oils.

26a (dr 82:18 by ¹fINMR): IR (neat) 2952, 2933, 1629, 1446, 1424, 1195cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.17-7.11 (m, 4 H), 4.81-4.80 (m, 1 H),4.25-4.24 (m, 1 H), 3.72 (s, 2 H), 3.46-3.40 (m, 1 H), 3.19 (s, 2 H),2.44 (brs, 4 H), 2.37 (s, 3 H), 2.31 (s, 3 H), 2.19-2.09 (m, 1 H),2.08-1.84 (m, 5 H), 1.80-1.66 (m, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ166.8, 164.8, 159.8, 141.9, 135.1, 130.5, 126.5, 126.2, 126.0, 109.9,55.8, 52.2, 39.2, 37.6, 32.5, 30.4, 28.9, 27.3, 23.8, 19.3, 11.0, 10.1;HRMS (ESI) m/z calcd for C₂₂H₂₉O₂N₂S ([M+H]⁺) 385.1950, found 385.1946.

26b (dr 92:8 by ¹HNMR): IR (neat) 2952, 2934, 1629, 1489, 1446, 1193,1163 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.20-7.12 (m, 4 H), 4.76 (t, 1 H,J=7.6 Hz), 4.20 (t, 1 H, J=7.6 Hz), 3.80 (d, 1 H, J=14.0 Hz), 3.62 (d, 1H, J=14.0 Hz), 3.20 (d, 1 H, J=12.8 Hz), 3.10 (d, 1 H, J=13.6 Hz),3.01-2.90 (m, 1 H), 2.60-2.45 (m, 5 H), 2.40 (s, 6 H), 2.22-2.11 (m, 1H), 2.10-2.00 (m, 1 H), 1.85-1.69 (m, 2 H), 1.55-1.40 (m, 2 H); ¹³C NMR(100 MHz, CDCl₃) δ 166.9, 166.3, 159.8, 142.0, 135.7, 130.4, 126.5,126.2, 126.0, 109.9, 53.3, 49.3, 39.0, 38.0, 32.8, 31.9, 31.1, 29.9,23.9, 19.5, 11.1, 10.2; HRMS (ESI) m/z calcd for C₂₂H₂₉O₂N₂S ([M+H]⁺)385.1950, found 385.1944.

-   (3,5-Dimethylisoxazol-4-yl)methanol (27) (Natale et al., Synth.    Commun. 1983, 13, 817-822.) To a solution of    3,5-dimethylisoxazole-4-carboxylic acid (1.60 g, 11.3 mmol) in THF    (69 mL) at 0° C. was added dropwise a 2 M solution of LiAlH₄ in THF    (5.6 mL, 11.2 mmol). The reaction mixture was allowed to warm to    room temperature, stirred overnight, transferred to a 500-mL    Erlenmeyer flask and treated with sodium sulfate decahydrate until    the foaming subsided. Celite (2.3 g) was added and the slurry was    filtered and washed with CH₂Cl₂ (75 mL). The filtrate was    concentrated in vacuo to give 27 (1.14 g, 8.97 mmol, 79%) as a clear    colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 4.46 (s, 2 H), 2.38 (s, 3    H), 2.29 (s, 3 H).

-   (3,5-Dimethylisoxazol-4-yl)methanethiol (25) (Moreno-Mañas et    al., J. Heterocycl. Chem. 1992, 29, 1557-1560.) A solution of    (3,5-dimethylisoxazol-4-yl)methanol (27, 0.500 g, 3.90 mmol) in    toluene (13 mL) was treated with Lawesson's reagent (0.890 g, 2.15    mmol) at room temperature, heated to 80° C. and stirred for 1 d. The    crude mixture was loaded directly onto SiO₂ and purified by    chromatography on SiO₂ (4:1, hexanes/EtOAc) to give 25 (0.115 g,    0.803 mmol, 21%) as a light yellow oil: ¹H NMR (300 MHz, CDCl₃) δ    3.49 (d, 2 H, J=6.6 Hz), 2.36 (s, 3 H), 2.30 (s, 3 H), 1.64 (t, 1 H,    J=6.6 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 165.2, 159.0, 113.3, 15.9,    10.9, 10.0.

-   Methyl 2-(phenylthio)acetate (28) (Bahrami et al., J. Org. Chem.    2010, 75, 6208-6213.) A solution of thiophenol (0.10 mL, 0.977    mmol), and methyl bromoacetate (0.164 g, 1.07 mmol) in THF (13 mL)    was treated with Et₃N (0.17 mL, 1.17 mmol), stirred at room    temperature for 4 h, and diluted with Et₂O and satd. aqueous NaHCO₃.    The aqueous layer was extracted with Et₂O (2×5 mL). The combined    organic layers were dried (MgSO₄), filtered and concentrated in    vacuo to give 28 (0.176 g, 0.966 mmol, 99%) as a clear oil: ¹H NMR    (300 MHz, CDCl₃) δ 7.42-7.38 (m, 2 H), 7.33-7.20 (m, 3 H), 3.71 (s,    3 H), 3.65 (s, 2 H); ¹³C NMR (75 MHz, CDCl₃) δ 170.1, 134.9, 129.9,    129.0, 127.0, 52.5, 36.5.

-   2-(Phenylthio)acetic acid (3d) (Bahrami et al., J. Org. Chem. 2010,    75, 6208-6213; Miura et al., Org. Lett. 2001, 3, 2591-2594.) To a    solution of methyl 2-(phenylthio)acetate (28, 0.176 g, 0.966 mmol)    in MeOH (2 mL) was added 2 M LiOH (1 mL). The reaction mixture was    stirred at room temperature for 1 h and TLC analysis (4:1,    hexanes/EtOAc) indicated that 28 had been consumed. The solution was    concentrated in vacuo, diluted with water (3 mL) and acidified to pH    2 with 1 M HCl at 0° C. The aqueous layer was extracted with EtOAc    (3×10 mL). The combined organic layers were dried (MgSO₄), filtered    and concentrated in vacuo to give 3d (0.144 g, 0.857 mmol, 89%) as a    colorless solid: ¹H NMR (300 MHz, CDCl₃) δ 11.27 (brs, 1 H), 7.43    (d, 2 H, J=7.6 Hz), 7.36-7.24 (m, 3 H), 3.69 (s, 2 H); ¹³C NMR (75    MHz, CDCl₃) δ 175.9, 134.4, 130.1, 129.2, 127.2, 36.6.

-   N,N′-Dimethyl-N-(o-tolyl)ethane-1,2-diamine (4i) (Gruseck et al.,    Tet. Lett. 1987, 28, 6027-6030.). A microwave vial was flushed with    argon and charged with the N,N′-dimethylethylene-diamine (0.180 g,    2.04 mmol), NaO-t-Bu (0.202 g, 2.04 mmol), (rac)-BINAP (0.0162 g,    0.0260 mmol), Pd₂(dba)₃ (0.0078 g, 0.0085 mmol), degassed toluene    (10.2 mL), and 2-bromotoluene (0.297 g, 1.70 mmol). The reaction    mixture was heated in the sealed vial under argon at 110° C. for 24    h, cooled to room temperature, diluted with CH₂Cl₂, filtered through    Celite, and concentrated in vacuo. The residue was purified by    chromatography on basic Al₂O₃ (95:5, CH₂Cl₂/MeOH) to give 4i (0.0508    g, 0.285 mmol, 17%) as a brown oil: ¹H NMR (400 MHz, CDCl₃) δ 7.16    (t, 2 H, J=7.6 Hz), 7.08 (d, 1 H, J=7.6 Hz), 6.98 (d, 1 H, J=7.2    Hz), 3.05 (t, 2 H, J=6.4 Hz), 2.71 (t, 2 H, J=6.4 Hz), 2.65 (s, 3    H), 2.43 (s, 3 H), 2.32 (s, 3 H), 1.36 (brs, 1 H); HRMS (ESI) m/z    calcd for C₁₁H₁₉N₂ ([M+H]⁺) 179.1543, found 179.1541.

-   1-(o-Tolyl)piperidin-4-ol (4n) (Harris et al., Org. Lett. 2002, 4,    2885-2888.) An oven-dried microwave tube was charged with Pd₂(dba)₃    (0.0606 g, 0.0653 mmol), CyJohnphos (0.0292 g, 0.0816 mmol), and    4-piperidinol (0.330 g, 3.26 mmol). The microwave tube was evacuated    and back-filled with argon. A 1 M solution of LiN(TMS)₂ (1.21 g,    7.17 mmol) in degassed THF (7.2 mL) was added via syringe along with    2-bromotoluene (0.600 g, 3.26 mmol). The reaction vessel was sealed    and heated at 65° C. with stirring for 22 h. The reaction mixture    was cooled to room temperature, quenched with 1 M HCl (10 mL),    stirred at room temperature for 5 min, neutralized with a satd.    aqueous NaHCO₃ solution, and diluted with EtOAc. The organic layer    was dried (MgSO₄), filtered through Celite, and concentrated in    vacuo. The crude residue was purified by chromatography on SiO₂    (ISCO, 12 g column, gradient hexanes to 3:7, EtOAc/hexanes) to give    4n (0.372 g, 1.94 mmol, 60%) as a brown oil: ₁H NMR (300 MHz, CDCl₃)    δ 7.17 (dd, 2 H, J=9.3, 7.2 Hz), 7.04-6.96 (m, 2 H), 3.87-3.81 (m, 1    H), 3.15-3.08 (m, 2 H), 2.74 (dt, 2 H, J=9.6, 2.7 Hz), 2.32 (s, 3    H), 2.06-2.00 (m, 2 H), 1.80-1.69 (m, 3 H); ¹³C NMR (75 MHz, CDCl₃)    δ 151.9, 132.7, 130.9, 126.4, 123.0, 119.0, 68.0, 49.8, 35.2, 17.7;    HRMS (ESI) m/z calcd for C₁₂H₁₈NO ([M+H]⁺) 192.1383, found 192.1307.

-   tert-Butyl 4-(o-tolyl)-1,4-diazepane-1-carboxylate (29a). A    microwave vial was flushed with argon and charged with    Boc-homopiperazine (0.223 g, 1.10 mmol), NaO-t-Bu (0.116 g, 1.20    mmol), (rac)-BINAP (0.0478 g, 0.0752 mmol, 7.5 mol %), Pd₂(dba)₃    (0.0233 g, 0.0251 mmol), degassed toluene (2.8 mL), and    2-bromotoluene (0.175 g, 1.00 mmol). The reaction mixture was heated    in the sealed vial under argon at 80° C. for 19 h, cooled to room    temperature, diluted with CH₂Cl₂, filtered through Celite, and    concentrated in vacuo. The residue was purified by chromatography on    SiO₂ (1:9, EtOAc/hexanes) to give 29a (0.139 g, 0.479 mmol, 48%) as    a yellow oil: IR (ATR) 2973, 2828, 1689, 1598, 1491, 1457, 1411,    1364, 1233, 1215, 1156, 1122, 878, 761, 725 cm⁻¹; ¹H NMR (500 MHz,    CDCl₃, room temperature, mixture of rotamers) δ 7.16 (d, 1 H, J=6.0    Hz), 7.12 (d, 1 H, J=6.0 Hz), 7.04 (d, 1 H, J=7.5 Hz), 6.95 (t, 1 H,    J=7.0 Hz), 3.61-3.56 (m, 4 H), 3.11-3.04 (m, 4 H), 2.31 (s, 3 H),    1.96-1.91 (m, 2 H), 1.49 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃, room    temperature, mixture of rotamers) δ 155.6, 155.5, 153.9, 153.8,    132.9, 130.9, 126.5, 123.1, 120.8 (2 C), 79.3, 56.2, 56.0, 55.5,    55.2, 48.4, 48.0, 46.2, 45.4, 29.0, 28.9, 28.5, 18.5; HRMS (ESI) m/z    calcd for C₁₇H₂₇N₂O₂ ([M+H]⁺) 291.2067, found 291.2062.

-   (3S,5R)-3,5-Dimethyl-1-(o-tolyl)piperazine (4k) (WO 2015/042297 A1).    A Schlenk flask was flushed with N₂ and charged with    cis-2,6-dimethylpiperazine (0.110 g, 0.963 mmol), NaO-t-Bu (0.170 g,    1.75 mmol), (rac)-BINAP (0.0084 g, 0.0130 mmol), Pd₂(dba)₃ (0.0083    g, 0.0087 mmol), degassed toluene (4 mL), and 2-bromotoluene (0.150    g, 0.880 mmol). The reaction mixture was heated under N₂ at 110° C.    for 24 h, cooled to room temperature, diluted with CH₂Cl₂, filtered    through Celite, and concentrated in vacuo. The crude residue was    purified by chromatography on SiO₂ (1:19, MeOH/CH₂Cl₂) to give 4k    (0.140 g, 0.685 mmol, 78%) as clear, yellow oil: ¹H NMR (500 MHz,    CDCl₃) δ 7.19-7.15 (m, 2 H), 7.02-6.98 (m, 2 H), 3.13-3.10 (m, 2 H),    3.01 (app d, 2 H, J=10.5 Hz), 2.35-2.31 (m, 5 H), 1.12 (d, 6 H,    J=6.5 Hz).

-   tert-Butyl (3R,5S)-3,5-dimethylpiperazine-1-carboxylate (30)    (Jacobsen et al., J. Med. Chem. 1999, 42, 1123-1144.) To a solution    of cis-2,6-dimethylpiperazine (0.500 g, 4.38 mmol) in CH₂Cl₂ (11 mL)    at 0° C. was added dropwise a solution of Boc-anhydride (0.946 g,    4.33 mmol) in CH₂Cl₂ (2.6 mL). The reaction mixture was allowed to    warm to room temperature, stirred overnight, diluted with CH₂Cl₂ and    washed with satd. aqueous Na₂CO₃ solution. The aqueous layer was    extracted with CH₂Cl₂. The combined organic layers were washed with    brine, dried (MgSO₄), filtered, and concentrated in vacuo to give 30    (0.813 g, 3.79 mmol, 87%) as an off-white solid: ¹H NMR (300 MHz,    CDCl₃) δ 4.10-3.80 (m, 2 H), 2.85-2.70 (m, 2 H), 2.40-2.20 (m, 2 H),    1.46 (s, 9 H), 1.05 (d, 6 H, J=6.3 Hz).

-   tert-Butyl (3R,5S)-3,5-dimethyl-4-phenylpiperazine-1-carboxylate    (29b). To a sealed tube under an argon atmosphere was added a    solution of KHMDS (0.241 g, 1.15 mmol) in dry 1,4-dioxane (2.0 mL),    a solution of tert-butyl 3,5-dimethylpiperazine-1-carboxylate (30,    0.246 g, 1.15 mmol) in dry 1,4-dioxane (0.9 mL) and bromobenzene    (100 μL, 0.955 mmol). The reaction mixture was stirred at 100° C.    for 18 h, cooled to room temperature, quenched with water (5 mL),    diluted with Et₂O (15 mL) and the aqueous layer was extracted with    Et₂O (2×15 mL). The combined organic layers were washed with brine,    dried (Na₂SO₄), filtered, and concentrated in vacuo. The crude    residue was purified by chromatography on SiO₂ (1:9, EtOAc/hexanes)    to give 29b (0.0970 g, 0.334 mmol, 35%) as a colorless oil: ¹H NMR    (300 MHz, CDCl₃) δ 7.33-7.27 (m, 2 H), 7.15-7.09 (m, 3 H), 4.00-3.80    (m, 2 H), 3.07-3.03 (m, 2 H), 2.82 (brt, 2 H, J=11.7 Hz), 1.50 (s, 9    H), 0.77 (d, 6 H, J=6.3 Hz).

-   tert-Butyl 3,5-dimethyl-4-(m-tolyl)piperazine-1-carboxylate (29c). A    sealed tube under an argon atmosphere was treated with KHMDS (0.221    g, 1.05 mmol) in dry 1,4-dioxane (2.0 mL), a solution of tert-butyl    3,5-dimethylpiperazine-1-carboxylate (30, 0.226 g, 1.05 mmol) in dry    1,4-dioxane (0.7 mL) and bromotoluene (105 μL, 0.877 mmol). The    reaction mixture was stirred at 100° C. for 18 h, cooled to room    temperature, quenched with water (5 mL), diluted with Et₂O (15 mL)    and the aqueous layer was extracted with Et₂O (2×15 mL). The    combined organic layers were washed with brine, dried (Na₂SO₄),    filtered, and concentrated in vacuo. The crude residue was purified    by chromatography on SiO₂ (1:9, EtOAc/hexanes) to give 29c (0.0441    g, 0.145 mmol, 17%) as a colorless oil: ¹H NMR (300 MHz, CDCl₃) δ    7.18 (t, 1 H, J=7.5 Hz), 6.96-6.89 (m, 3 H), 4.00-3.80 (m, 2 H),    3.06-3.00 (m, 2 H), 2.81 (brt, 2 H, J=11.7 Hz), 2.32 (s, 3 H), 1.50    (s, 9 H), 0.77 (d, 6 H, J=6.3 Hz).

Example 2 Synthesis and Characterization of(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)((1RS,2SR)-2-(4-fluorophenyl)cyclopropyl)methanone(JJ-450)

-   ((4-Fluorophenyl)ethynyl)trimethylsilane (Everett et al., Org. Lett.    2013, 15, 2926-2929; Yonemoto-Kobayashi et al., Org. Biomol. Chem.    2013, 11, 3773-3775). A flame-dried flask under Ar was charged with    Pd(PPh)₂Cl₂ (0.361 g, 0.514 mmol), CuI (0.0979 g, 0.514 mmol), and    4-fluorobromobenzene (5.66 mL, 51.4 mmol). Et₃N (110 mL) and    (trimethylsilyl)acetylene (10.9 mL, 77.1 mmol) were added via    syringe and the solution was sparged with Ar for 30 min. The    reaction mixture was heated to 80° C. overnight and analysis by TLC    (4:1, hexanes/EtOAc) indicated that 4-fluorobromobenzene had been    consumed. The solution was cooled to room temperature and filtered    through celite. The celite was washed (Et₂O) until the washes    appeared colorless. The combined filtrates were concentrated in    vacuo. The crude residue was purified by chromatography on SiO₂    (hexanes) to afford 4-fluorophenyl)ethynyl)trimethylsilane (9.03 g,    47.0 mmol, 91%) as a pale orange oil: ¹1-1 NMR (300 MHz, CDCl₃) δ    7.47-7.42 (m, 2 H), 6.99 (t, 2 H, J=8.7 Hz), 0.25 (s, 9 H); ¹³C NMR    (75 MHz, CDCl₃) δ 162.6 (d, J_(C F)=248 Hz), 133.9 (d, J_(C—F)=8    Hz), 119.3 (d, J_(C—F)=4 Hz), 115.5 (d, J_(C—F)=22 Hz), 104.0, 93.8,    −0.07.

-   3-(4-Fluorophenyl)propiolic acid (Yonemoto-Kobayashi et al., Org.    Biomol. Chem. 2013, 11, 3773-3775). CsF (4.74 g, 31.2 mmol) was    loaded into an oven-dried 250-mL round bottom flask in a glovebox.    The flask was removed from the glovebox, attached to a CO₂ balloon,    equipped with a magnetic stirrer and a septum, and filled with    anhydrous DMSO (60 mL). Neat    ((4-fluorophenyl)ethynyl)trimethylsilane (5.00 g, 26.0 mmol) was    added dropwise. The reaction mixture was stirred under CO₂ at room    temperature overnight, diluted with water (600 mL) and washed with    CH₂Cl₂ (2×150 mL). The aqueous layer was acidified at 0° C. to pH 1    with 6 M HCl and then extracted with Et₂O (3×200 mL). The combined    organic layers were dried (MgSO₄), filtered, and concentrated in    vacuo to afford 3-(4-fluorophenyl)propiolic acid (3.02 g, 18.4 mmol,    71%) as an orange solid: ¹H NMR (400 MHz, Acetone-d₆) δ 11.74 (brs,    1 H), 7.71 (dd, 2 H, J=8.6, 5.6 Hz), 7.26 (t, 2 H, J=8.6 Hz); ¹³C    NMR (100 MHz, Acetone-d₆) δ 164.8 (d, J_(C—F)=249 Hz), 154.7, 136.1    (d, J_(C—F)=9 Hz), 117.1 (d, J_(C—F)=23 Hz), 84.6, 81.8.

-   1-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-yn-1-one.    To a solution of 3-(4-fluorophenyl)propiolic acid (3.00 g, 18.3    mmol) in anhydrous CH₂Cl₂ (180 mL) at 0° C. was added    1-(5-chloro-2-methylphenyl)piperazine (4.62 g, 21.9 mmol), and Et₃N    (6.35 mL, 45.7 mmol), followed by dropwise addition of T3P (50 wt. %    solution in EtOAc, 19.4 mL, 27.4 mmol). The reaction mixture was    stirred at 0° C. for 30 min, warmed to room temperature overnight,    diluted with CH₂Cl₂ (200 mL), washed with 1 M HCl (150 mL), dried    (MgSO₄), filtered, and concentrated in vacuo. The residue was    purified by chromatography on SiO₂ (2:1, hexanes/EtOAc) to give    1-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-yn-1-one    (5.22 g, 14.6 mmol, 80%) as an off white solid: Mp 138.7-140.4° C.;    IR (neat) 2924, 2216, 1625, 1596, 1504, 1443, 1431, 1219, 1038, 837    cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.55 (dd, 2 H, J=7.5, 5.4 Hz),    7.12-6.94 (m, 5 H), 3.96 (app t, 2 H, J=4.8 Hz), 3.82 (app t, 2 H,    J=4.8 Hz), 2.95 (app t, 2 H, J=4.8 Hz), 2.87 (app t, 2 H, J=4.8 Hz),    2.28 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 163.5 (d, J_(C—F)=251 Hz),    153.0, 151.7, 134.5 (d, J_(C—F)=9 Hz), 132.1, 131.8, 130.9, 123.7,    119.8, 116.4 (d, J_(C—F)=4 Hz), 116.0 (d, J_(C—F)=23 Hz), 89.9,    80.9, 51.9, 51.3, 47.4, 41.8, 17.3; HRMS (ESI) m/z calcd for    C₂₀H₁₉ClFON₂ ([M+H]⁺) 357.1164, found 357.1165.

-   (Z)-1-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-en-1-one.    To a solution of    1-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-yn-1-one    (5.00 g, 14.0 mmol) in dry EtOAc (140 mL) was added Lindlar's    catalyst (5% Pd on CaCO₃, lead poisoned, 0.298 g, equivalent to 1    mol % Pd) and quinoline (0.83 mL, 7.01 mmol). The reaction vessel    was placed under vacuum, backfilled with H₂ (balloon, 2×) and    allowed to stir at room temperature for 6 h. Analysis by TLC (2:1,    hexanes/EtOAc) indicated that    1-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-yn-1-one    had been mostly consumed. The reaction mixture was filtered through    Celite, washed with EtOAc, and concentrated under vacuum. The    combined organic layers were washed with 1 M HCl, dried (MgSO₄),    filtered, and concentrated in vacuo. The crude material was purified    by chromatography on SiO₂ (1:1, hexanes/EtOAc) to afford    (Z)-1-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-en-1-one    (3.15 g, 8.78 mmol, 63%, 87% brsm) as a colorless solid: IR (neat)    2913, 2239, 1616, 1506, 1437, 1223, 837, 725 cm⁻¹; ¹H NMR (400 MHz,    CDCl₃) δ 7.41-7.36 (m, 2 H), 7.08-7.02 (m, 3 H), 6.96 (dd, 1 H,    J=8.1, 2.1 Hz), 6.80 (d, 1 H, J=2.1 Hz), 6.66 (d, 1 H J=12.5 Hz),    6.05 (d, 1 H, J=12.5 Hz), 3.80 (m, 2 H, J=5.0 Hz), 3.49 (t, 2 H,    J=5.0 Hz), 2.80 (t, 2 H, J=5.0 Hz), 2.53 (t, 2 H, J=5.0 Hz), 2.21    (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 162.7 (d, J_(C—F)=248    Hz), 151.7, 132.6, 132.0, 131.8, 131.5 (d, J_(C—F)=3 Hz), 132.1,    131.8, 130.9, 130.2 (d, J_(C—F)=8 Hz), 123.6, 122.7, 119.6, 115.6    (d, J_(C—F)=21 Hz), 51.4, 51.2, 46.5, 41.5, 17.3; HRMS (ESI) m/z    calcd for C₂₀H₂₁ClFON₂ ([M+H]⁺) 359.1321, found 359.1329.

-   (4-(5-Chloro-2-methylphenyl)piperazin-1-yl)((1RS,2SR)-2-(4-fluorophenyl)cyclopropyl)-methanone    (JJ-450). THF (90 mL) was degassed by sparging with Ar for 60 min    and treated at room temperature under Ar atmosphere with anhydrous    CrCl₂ (6.43 g, 51.8 mmol) followed by    (Z)-1-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-3-(4-fluorophenyl)prop-2-en-1-one    (3.10 g, 8.64 mmol) and CH₂ICl (3.36 mL, 43.2 mmol). The reaction    mixture was stirred for 20 h at 80° C., cooled to room temperature,    quenched by the addition of 1.0 M aqueous HCl (300 mL) and extracted    with EtOAc (3×300 mL). The combined organic layers were filtered    through a plug of basic Al₂O₃, and concentrated in vacuo. The    residue was purified by chromatography on SiO₂ (1:1, hexanes/EtOAc)    to afford an oil that was further purified twice by chromatography    on basic Al₂O₃ (1:1, hexanes/EtOAc) to give    (4-(5-chloro-2-methylphenyl)piperazin-1-yl)((1RS,2SR)-2-(4-fluorophenyl)cyclopropyl)methanone    (2.76 g, 7.41 mmol, 86%) as a clear oil that solidified after    storage on high vacuum overnight: Mp 78.2-80.4° C. (hexanes); IR    (CH₂Cl₂) 2936, 1637, 1592, 1510, 1487, 1435, 1223, 1033, 837, 815    cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.16-7.11 (m, 2 H), 7.07 (dd, 1 H,    J=8.1, 0.5 Hz), 7.00-6.94 (m, 3 H), 6.73 (d, 1 H, J=2.1 Hz),    3.81-3.76 (m, 1 H), 3.71-3.60 (m, 2 H), 3.36 (ddd, 1 H, J=12.4, 8.8,    3.1 Hz), 2.79-2.71 (m, 2 H), 2.45 (td, 1 H, J=8.8, 7.0 Hz),    2.35-2.29 (m, 1 H), 2.26-2.16 (m, 5 H), 1.83 (dt, 1 H, J=7.0, 5.6    Hz), 1.35 (td, 1 H, J=8.8, 5.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ    167.2, 161.7 (d, J_(C—F)=244 Hz), 151.9, 133.1 (d, J_(C—F)=3 Hz),    131.9 (d, J_(C—F)=14 Hz), 130.9, 129.1 (d, J_(C—F)=8 Hz), 123.6,    119.7, 115.0 (d, J_(C—F)=21 Hz), 51.8, 51.6, 45.6, 42.2, 23.8, 23.5,    17.3, 10.7; HRMS (ESI) m/z calcd for C₂₁H₂₃ClFON₂ ([M+H]⁺) 373.1477,    found 373.1478.

Racemic JJ-450 was separated on a SFC Chiralpak-IC semiprep (250×10 mm)column (20% MeOH, 6 mL/min, 220 nM, P=100) to afford(4-(5-chloro-2-methylphenyl)piperazin-1-yl)((1S,2R)-2-(4-fluorophenyl)cyclopropyl)methanoneJJ-450A (retention time 13.1 min) as a colorless viscous oil (100%purity by ELSD): [α]²⁰ _(D) −118.7 (c 0.39, CHCl₃); ¹H NMR (300 MHz,CDCl₃) δ 7.17-7.10 (m, 2 H), 7.07 (d, 1 H, J=8.1 Hz), 7.02-6.94 (m, 3H), 6.72 (d, 1 H J=2.1 Hz), 3.83-3.75 (m, 1 H), 3.72-3.58 (m, 2 H),3.39-3.31 (m, 1 H), 2.81-2.69 (m, 2 H), 2.45 (td, 1 H, J=8.7, 6.9 Hz),2.36-2.25 (m, 1 H), 2.25-2.15 (m, 5 H), 1.83 (dt, 1 H, J=6.9, 5.5 Hz),1.35 (td, 1 H, J=8.7, 5.5 Hz); HRMS (ESI) m/z calcd for C₂₁H₂₃ClFON₂([M+H]⁺) 373.1477, found 373.1476. The enantiomeric excess was 100% ee(SFC Chiralpak-IC (250×4.6 mm); 20% MeOH, 220 nM, 2 mL/min; retentiontime: 9.8 min).

(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)((1R,2S)-2-(4-fluorophenyl)cyclopropyl)-methanoneJJ-450B (retention time 16.5 min) was obtained as a colorless viscousoil (100% purity by ELSD): [α]²⁰ _(D)+117.4 (c 0.38, CHCl₃); ¹H NMR (300MHz, CDCl₃) δ 7.17-7.10 (m, 2 H), 7.07 (d, 1 H, J=8.1Hz), 7.01-6.94 (m,3 H), 6.72 (d, 1 H, J=2.1 Hz), 3.82-3.74 (m, 1 H), 3.71-3.60 (m, 2 H),3.39-3.30 (m, 1 H), 2.81-2.68 (m, 2 H), 2.45 (td, 1 H, J=8.6, 7.0 Hz),2.35-2.26 (m, 1 H), 2.25-2.15 (m, 5 H), 1.83 (dt, 1 H, J=7.0, 5.6 Hz),1.35 (td, 1 H, J=8.6, 5.6 Hz); HRMS (ESI) m/z calcd for C₂₁H₂₃ClFON₂([M+H]⁺) 373.1477, found 373.1476. The enantiomeric excess was 100% ee(SFC Chiralpak-IC (250×4.6 mm); 20% MeOH, 220 nM, 2 mL/min; retentiontime: 12 min).

Example 3 Activity of Compounds in PSA Luciferase Assay

The biological activity of analogs 5-16, 18, 20, 26, JJ-450, and theresolved enantiomers JJ-450A and J-450B was determined and compared toHTS hit 1 (IC₅₀ 7.3 μM) and MDV3100 (IC₅₀ 1.1 μM) using the Dual-Gloluciferase system (Promega, WI, USA) in C4-2-PSA-rl cells, which weregenerated by transfection with PSA6.1-luc and pRL-TK followed by stableselection using G418 and puromycin. C4-2-PSA-rl stable cells werecultured in RPMI 1640 medium with 10% FBS, 1% penicillin-streptomycin,1% L-glutamine, 10 mg/mL puromycin, and 50 mg/mL G418. C4-2-PSA-rl cellswere seeded in 24-well plates such that they reached 75-80% cellmonolayer density after 24 h. C4-2-PSA-rl cells were then treated for 24h with 0, 0.2, 0.8, 3.2, 12.8, or 25 μM of each compound dissolved inDMSO (0.8% DMSO/well) in the presence of 1 nM synthetic androgen R1881,with each experimental condition in triplicate. The cells were alsotreated in parallel with 12.8 μM compound 1 and 12.8 μM MDV3100 aspositive controls. Each compound was tested in at least two independentexperiments. Luciferase activity was assayed using the Dual-Luciferase®Reporter Assay System (Promega) using LMax II Microplate Reader(Molecular Devices). The luciferase assay results were acquired usingSoftMax Pro5.45 software (Molecular Devices) and analyzed using GraphPadPrism. PSA6.1-luc activity was normalized to the Renilla luciferaseactivity. Relative luciferase activity was calculated as the quotient ofandrogen-induced PSA-firefly/Renilla luciferase activity. Since PSApromoter activity correlates to AR transcriptional activity, inhibitionof AR will result in decreased PSA-luciferase activity. IC₅₀ values werecalculated using GraphPad Prism and data represent the mean and SD of2-6 independent experiments (Table 2).

TABLE 2 In vitro activity of analogs in the PSA luciferase assay inC4-2-PSA-rl cells. Entry Compound IC₅₀ (μM) 1  1  7.3 ± 2.5^(c) 2  5a>25^(a) 3  5b 14.5 ± 3.2^(b) 4  5c >25^(a) 5  5d >25^(a) 6  5e 12.0 ±1.6^(b) 7  5f 12.6 ± 7.7^(b) 8  5g 11.1 ± 5.3^(b) 9  5h >25^(a) 10  5i18.4 ± 9.2^(b) 11  5j 11.1 ± 3.3^(a) 12  5k  3.1 ± 1.1^(a) 13  5l 14.7 ±4.4^(a) 14  5m 16.6 ± 4.8^(b) 15  6 10.8 ± 5.7^(b) 16  7 13.7 ± 0.8^(b)17  8 14.4 ± 3.7^(b) 18  9 >25^(a) 19 10 20.3 ± 11.6^(a) 20 11 >25^(a)21 12 >25^(b) 22 13 16.1 ± 3.3^(b) 23 14 12.7 ± 0.8^(a) 24 15  2.9 ±1.0^(b) 25 16 >25^(b) 26 18a >25^(b) 27 18b >25^(b) 28 18c 7.2 ± 2.7^(c)29 20a >25^(a) 30 20b >25^(c) 31 26a  7.7 ± 1.6^(b) 32 26b  7.9 ±2.8^(a) 33 JJ-450  2.7 ± 1.1 34 JJ-450A  1.6 ± 0.1 35 JJ-450B 13.1 ± 1.836 MDV3100  1.1 ± 0.5^(e) Assay repeats: ^(a)n = 2; ^(b)n = 3; ^(c)n =4; ^(d)n = 5; ^(e)n = 6.

Modifications of the substituents on the benzene ring in zone 1 revealedthat methyl groups in the 3- and 4-positions (5c, 5d) led to loss ofactivity, while the 2-methyl analog 5b (IC₅₀ 14.5 μM) retained abouthalf of the activity of the 2,3-dimethylated 1 (Table 2). Removal of the2-methyl group in 5a deleted activity. In agreement with this trend inzone 1, the bulky 1-naphthyl substituent (5g) recovered activity (IC₅₀11.1 μM). Analogs with electron-withdrawing substituents at the benzene2-position (2-NC, 5e, and 2-F, 5f) also maintained or slightly increasedactivity (IC₅₀ 12-13 μM); however, the electron-donating 2-methoxysubstituted 5h was not tolerated and resulted in a complete loss ofactivity, possibly due to an increase in the pKa of the aniline and/oran unfavorable increase in the t-electron density of the aromatic ring.

The piperazine core (zone 2) was queried through substitutions withflexible as well as constrained acyclic and cyclic diamines The flexibleN,N′-dimethylethylenediamine linker in 5i (IC₅₀ 18.4 μM) and the7-membered diazepane 5j (IC₅₀ 11.1 μM) both conserved activity. Thedimethylated piperazines 51 and 5m (IC₅₀ 15-17 μM) were both also almostas active as the initial hit. In contrast, the conformationally morehighly constraint 2,6-dimethylpiperazine 5k was considerably more activewith an IC₅₀ of 3.1 μM. Installment of an ethylene bridge and acarbon-linked (2-Me)Ph group decreased activity again, since bothdiastereomers of the bicyclo[3.2.1] ring systems 26a and 26b, showed anIC₅₀ of 8 μM.

Reduction of amide 5b to amine 6 resulted in a 1.3-fold increase inactivity to an IC₅₀ of 10.8 μM. Sulfonamide 18c (IC₅₀ 7.2 μM) was twiceas active as the initial hit 1, but urea 20a and carbamate 20b wereinactive.

The replacement of the thioether linkage in zone 2 with an ether groupabolished activity in 18a. Substituting the thioether with theN-methylated amine in 18b also abolished activity. In contrast, in ananalogous system with a phenyl group in place of the isoxazole, boththioether 7 as well as the all-carbon chain containing 8 showed constantactivity (IC₅₀ 14 μM).

In order to verify that the biological effect in the thioether serieswas not a result of S-oxidation in the cellular assay, common productsof thioether oxidation, i.e. sulfoxide 12 and sulfone 13, were tested.While sulfone 13 retained some activity (IC₅₀ 16.1 μM), sulfoxide 12 wasinactive. Shortening the three-atom chain to afford the two-atomthioether-linked 9 also abolished activity. The rigidified alkyne 10 andthe corresponding (E)-alkene 11 and its cyclopropane isostere 16 werealso found to be essentially inactive. In contrast, the (Z)-alkene 14surprisingly showed an IC₅₀ of 12.7 μM, and the corresponding cis-fusedcyclopropane isostere 15 was even more potent than analog 1, showing anIC₅₀ of 2.9 μM (Table 2).

In summary, zone 1 modifications showed that the ortho-substituent onthe phenyl ring was important for activity. In zone 2, the stericallyencumbered 2,6-dimethylpiperazine proved superior to flexible,unsubstituted, and bridged analogs. In zone 3, a carbonyl group was notrequired, and a sulfonamide and even the reduced amine were welltolerated. In zone 4, thioether oxidation reduces activity, and only thecis-cyclopropane successfully and significantly improves the IC₅₀.Limited substitutions were performed in zone 5, but in general analogswith a phenyl group were equipotent with their 3,5-dimethylisoxazolecongeners (see, for example, 7 vs 5b). Compounds 5k, 15, and JJ-450(particularly JJ-450A) were found to be significantly more potentthan 1. Compounds 15 and JJ-450 are of particular interest due to theisosteric replacement of the thioether linker with the metabolicallymore stable cyclopropane.

Compounds 559, 562, 475, 476, 484, and 458 are all also active in thePSA luciferase assay at sub-micromolar EC₅₀s (450-900 nM), and they areinactive against androgen receptor (AR) negative cell lines in cellproliferation assays.

Additional compounds are shown below:

Compound #583 is very potent, with an IC₅₀>1 uM in inhibitingAR-dependent PSA promoter activity (FIG. 9A). As expected, #583inhibited proliferation of AR-positive C4-2 (FIG. 9B), but notAR-negative PC3 (FIG. 9C), prostate cancer cells. Also, #583 does notcontain a sulfur atom in the structure and should therefore be moreresistant to oxidative metabolic degradation than the sulfur-containingcompounds.

Compounds #571 and #425 were developed for conjugation to agarosematrix. #571 is quite active, with an IC₅₀ of ˜3 uM in the inhibition ofAR activation of PSA promoter in a luciferase assay (FIG. 10).

Example 4 Inhibition of Xenograft Tumor Growth by JJ-450

22Rv 1 xenograft tumors were established in SCID mice by subcutaneousinjection of 2×10⁶ cells in Matrigel. Once the tumors reached ˜150 μL involume, the mice were castrated and randomized into 3 groups for dailyIP injection of vehicle (n=11), 10 mg/kg (n=11) and 75 mg/kg (n=11)groups. Injection of JJ-450 was initiated at time of castration. Tumorvolumes were measured 3 times every week. As shown in FIG. 11, compoundJJ-450 significantly inhibited tumor growth. Error bars, SEM.

LNCaP xenograft tumors were established in SCID mice by subcutaneousinjection of 2×10⁶ cells in Matrigel. Once the tumors reached ˜200 ul involume, the mice were castrated and randomized into 4 groups: oralgavage of vehicle (n=6), oral gavage at 10 mg/kg (n=6), IP injection at10 mg/kg (n=8), and oral gavage at 75 mg/kg (n=7) groups. Administrationof JJ-450 was started 2 weeks after castration. Tumor volumes weremeasured twice every week. As shown in FIG. 12, compound JJ-450significantly inhibited tumor growth. Error bars, SEM.

In view of the many possible embodiments to which the principles of thedisclosed compounds, compositions and methods may be applied, it shouldbe recognized that the illustrated embodiments are only preferredexamples and should not be taken as limiting the scope of the invention.

What is claimed is:
 1. A method for treating prostate cancer in asubject, comprising administering to the subject a therapeuticallyeffective amount of a compound, or a pharmaceutically acceptable salt orester thereof, selected from:


2. The method of claim 1, wherein the compound is:


3. The method of claim 1, wherein the compound is:


4. The method of claim 1, wherein the compound is:


5. The method of claim 1, wherein the compound is:


6. The method of claim 1, wherein administering a therapeuticallyeffective amount of the compound comprises administering apharmaceutical composition comprising the therapeutically effectiveamount of the compound and at least one pharmaceutically acceptableadditive.
 7. The method of claim 1, wherein the prostate cancer iscastration-resistant prostate cancer.
 8. The method of claim 1, whereinthe compound is orally administered.
 9. The method of claim 1, whereinthe method is used in combination with androgen deprivation therapy. 10.The method of claim 1, wherein the agent is co-administered withabiraterone.
 11. The method of claim 1, wherein the agent isco-administered with enzalutamide.
 12. The method of claim 1, whereinthe method further comprises identifying a subject that is in need oftreatment with the agent.
 13. The method of claim 1, wherein thetherapeutically effective amount of the compound is from about 0.01mg/kg body weight to about 20 mg/kg body weight.
 14. The method of claim1, wherein administering the therapeutically effective amount of thecompound reduces a nuclear level of androgen receptor incastration-resistant prostate cancer (CRPC) cells relative to untreatedCRPC cells.
 15. The method of claim 14, wherein reducing the nuclearlevel of androgen receptor inhibits activation of the androgen receptor.16. The method of claim 15, wherein a reduction in androgen receptoractivation is determined by measuring prostate-specific antigen.
 17. Amethod for inhibiting proliferation of prostate cancer cells, the methodcomprising: contacting prostate cancer cells with an effective amount ofa compound, or a pharmaceutically acceptable salt or ester thereof,selected from:

thereby inhibiting proliferation of the prostate cancer cells.
 18. Themethod of claim 17, wherein the prostate cancer cells areandrogen-dependent cells.
 19. A method for reducing a nuclear level ofandrogen receptor is castration-resistant prostate cancer cells, themethod comprising: contacting castration-resistant prostate cancer(CRPC) cells with an effective amount of a compound, or apharmaceutically acceptable salt or ester thereof, selected from:

thereby reducing a nuclear level of androgen receptor in the CRPC cells.20. The method of claim 19, further comprising: determining reduction ofthe nuclear level of androgen receptor by measuring prostate-specificantigen.